Chemical synthesis using solvent microdroplets

ABSTRACT

The present invention relates to microdroplets of a solution comprising a solvent having a boiling point of 150° C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec). Such microdroplets are useful for the synthesis of chemical species, particularly biopolymers such as oligonucleotides and peptides, as well as arrays of chemical species. Preferably, the solvent has the formula (I):  
                 
 
     wherein  
     A=O or S;  
     X, O, S or N(C 1 -C 4  alkyl);  
     Y=O, S, N(C 1 -C 4  alkyl) or CH 2 ; and  
     R=C 1 -C 20  straight or branched chain alkyl.

[0001] This invention was made with government support under grantnumber BIR92-14821 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to chemical synthesis, particularlysynthesis of biopolymers such as oligonucleotides and peptides, usingsolvent microdroplets as a means for reagent delivery.

BACKGROUND OF THE INVENTION

[0003] Genetic information generated by the Human Genome Project isallowing scientists, physicians, and others to conduct diagnostic andexperimental procedures on an unprecedented scale in terms of speed,efficiency, and number of screenings performed within one procedure. Inorder to make full use of this new information, there is an urgent needfor the ability to screen a large number of chemical compounds,particularly oligonucleotide probes, against samples of DNA or RNA fromnormal or diseased cells and tissue. One important tool for suchanalyses is nucleic acid hybridization, which relies on the differencein interaction energies between complementary and mismatched nucleicacid strands (see U.S. Pat. No. 5,552,270 to Khrapko et al.). Using thistool, it is possible to determine whether two short pieces of nucleicacid are exactly complementary. Longer nucleic acids can also becompared for similarity.

[0004] Nucleic acid hybridization is often used for screening clonedlibraries to identify similar, and thus presumably related, clones. Thisprocedure typically involves using (a) natural nucleic acid targetswhich are usually bound to a membrane, and (b) a natural or syntheticnucleic acid probe which is washed over many targets at once. With theappropriate mechanics, membranes can be constructed with targets at adensity of generally between one and ten targets per mm². Hybridizationdetection is carried out by labeling the probe, for example eitherradioactively or with chemiluminescent reagents, and then recording theprobe's emissions onto film.

[0005] Alternative approaches to nucleic acid hybridization haveinvolved oligonucleotide probes that are synthesized on a solid supportor a substrate, and then hybridized to a single natural target. Solidphase synthesis techniques for obtaining peptides (K. S. Lam et al.,Nature 354:82 (1991) and Geysen et al., J. Immunol. Methods 102:259(1987)) and oligonucleotides (J. Weiler et al., Anal. Biochem. 243:218(1996) and U. Maskos et al., Nucleic Acids Res. 20(7):1679 (1992); T.Atkinson et al., Solid-Phase Synthesis of Oligodeoxyribonucleotides bythe Phosphitetriester Method, in Oligonucleotide Synthesis 35 (M. J.Gait ed., 1984) have been disclosed. While such approaches have thepotential for large-scale assembly of oligonucleotide arrays, the costof making such a variety of arrays is prohibitive.

[0006] Recently, there have been reports of using microdrop dispensersto generate oligomers and polymers arranged, on a substrate, in arraysof microdroplets:

[0007] 1. T. Brennan, Human Genome Program, U.S. Department of Energy,Contractor-Grantee Workshop III, February 7-10, 1993, Santa Fe, N. Mex.,Methods to Generate Large Arrays of Oligonucleotides 92 (1993),discloses that arrays of oligonucleotides were sought to be synthesizedin parallel chemical reactions on glass plates, using arrays ofpiezoelectric pumps, similar to an inkjet printer, as a means fordelivering reagents. In such a scheme, each array element is separatedby its neighbor by a perfluoroalkane tension barrier which is not wet bythe acetonitrile reaction solvent.

[0008] 2. U.S. Pat. No. 5,449,754 to Nishioka discloses that peptidearrays can be obtained using an inkjet print head to deposit adimethylformamide solution of N-protected activated amino acids, in theform of microdroplets, onto an aminosilylated glass slide which issubsequently washed with a trifluoroacetic acid solution to remove theN-protecting groups from the anchored amino acids. The process isrepeated until amino acids having the desired sequence are obtained.

[0009] 3. U.S. Pat. No. 5,474,796 to Brennan describes a piezoelectricimpulse jet pump apparatus for synthesizing arrays of oligomers orpolymers having subunits connected by ester or amide bonds. According tothat scheme, a glass plate is coated with a fluoropolymer which is thenselectively removed, leaving glass regions, in spots upon which oligomeror polymer synthesis would take place. The glass regions are epoxidizedand subsequently hydrolyzed to afford a hydroxyalkyl group that wouldreact with an activated chemical species. Where the oligomers sought tobe synthesized are oligonucleotides, microdroplets of acetonitrile ordiethyleneglycol dimethyl ether solutions of 5′-protected nucleotidemonomers that are activated at their 3′-positions would be dispensed viaa piezoelectric jet head, and would impinge upon the hydroxyalkyl group,forming a covalent bond therewith. After removing the 5′-protectinggroups by flooding the surface of the plate with a deprotecting reagent,the process is repeated until the desired oligonucleotides are obtained.

[0010] 4. International Publication No. WO 95/25116 by Baldeschwieler etal. discloses a method for chemical synthesis at different sites on asubstrate using an inkjet printing device to deliver reagents tospecific sites of the substrate. In that instance, the inkjet printingdevice would deposit, in sequence, (a) a protected molecule onto thesubstrate, (b) a deprotecting reagent onto the protected molecule so asto expose a reactive site, and (c) a second protected molecule at thesite of the now-deprotected molecule, so as to form a growing chain ofmolecules. The entire process is repeated as necessary. According tothis publication by Baldeschwieler et al., useful reaction solvents aredibromomethane, nitromethane, acetonitrile and dimethylformamide.

[0011] 5. U.S. Pat. No. 5,658,802 to Hayes et al. discloses a dispensingapparatus that is allegedly capable of providing droplets having avolume of 10 pL to 100 pL, and purportedly useful for synthesizingarrays of diagnostic probes. According to that reference, the dispensingapparatus is capable of dispensing “liquids” that may contain DNAmolecules, peptides, antibodies, antigens, enzymes or entire cells;however, no specific examples of such “liquids” are disclosed.

[0012] There exists a need for a method of efficiently synthesizingchemical compounds on a large scale that can be automated. Prior artsuggestions for achieving such involve various drawbacks.

[0013] The present inventor has realized the nature of these drawbacks,which is overcome by the present invention. In particular, thedispensation of certain organic solvents from an inkjet printing devicefor use in chemical synthesis has several drawbacks. First, many organicsolvents, such as alcohols or amines, bear functional groups that arecapable of reacting with those chemical compounds sought to be dispensedfrom the inkjet device. Second, solvents having boiling points of lessthan 150° C. are relatively volatile, and can evaporate from a substratebefore the reactant(s) dissolved therein have completely reacted withany species bound to the substrate. Third, such volatile solvents canbegin to evaporate at the site of the inkjet print head, causingreactants dissolved in the solvents to precipitate and clog the inkjetnozzle. Fourth, solvents that have surface tension values that are lowerthan 30 dynes/cm at room temperature have a relatively high affinity forthe face of the inkjet nozzle, and tend to give rise to unstable andnon-uniformly sized droplets. Fifth, solvents that have viscosity valuesthat are lower than 1 centipoise at room temperature tend to formnon-uniformly sized droplets due to their response to residualoscillations in the solvent. Sixth, many organic solvents, particularlyacetonitrile, have the highly undesirable characteristic of beingcapable of dissolving adhesives and plastics used in inkjet print heads.Thus, prior to the present invention the organic solvents used forsynthesizing oligonucleotides were ineffective in automated systemsemploying plastic components such as ink jet print heads.

[0014] Thus, there exists a need for a class of organic solvents, usefulfor chemical synthesis, that is relatively inert, and that has boilingpoint, surface tension and viscosity properties that are optimal formicrodroplet formation from an inkjet device. Such a need is satisfiedby the present invention.

[0015] The use of inkjet printing technology in chemical synthesis wouldbe particularly useful for a large-scale synthesis of biopolymers, suchas oligonucleotides. While a manual approach might improve theefficiency of large-scale synthesis to some degree, manual steps wouldbe time-consuming. Specifically, a rinsing step would be performed aftereach deposition step to rinse away the unattached monomers, which wouldbe time-consuming if done manually.

[0016] The present inventor has also appreciated that in order toalternate efficiently between the deposition step and the rinsing step,a system may be designed in such a way that the substrate is made tomove while the print heads remain stationary, depositing microdropletsof nucleoside monomers. However, each time the substrate is positionedfor deposition, the substrate must be aligned correctly relative to theprint heads to ensure that the monomers can be deposited at preciselocations on the substrate. This is a time-consuming process if it is tobe done manually.

[0017] Thus, there exists a need for an automated system for efficientlyperforming large-scale synthesis of biopolymers using inkjet printingtechnology and particularly a need for an automated alignment mechanismwhich can be used to position the substrate precisely with respect tothe print heads without manual intervention. Such a need is satisfied bythe present invention.

[0018] Citation of any references above shall not be construed as anadmission that such reference is available as prior art to the presentapplication.

SUMMARY OF THE INVENTION

[0019] The present invention relates to a microdroplet of a solution,the solution comprising a solvent having a boiling point of 150° C. orabove, a surface tension of 30 dynes/cm or above, and a viscosity of0.015 g/(cm)(sec) or above.

[0020] The invention further provides a method for dispensingmicrodroplets of a solution from a microdroplet dispensing device, themicrodroplet dispensing device comprising (a) a manifold which containsthe solution, (b) a nozzle at one end of the manifold and (c) means forapplying a pressure pulse to the manifold, the means located at theother end of the manifold, comprising the step of applying a pressurepulse to the manifold, thereby dispensing the solution through thenozzle in microdroplet form, the solution comprising a solvent having aboiling point of 150° C. or above, a surface tension of 30 dynes/cm orabove, and a viscosity of 0.015 g/(cm)(sec) or above.

[0021] The invention still further provides a method for chemicalsynthesis, comprising the step of dispensing a microdroplet of asolution comprising (i) a first chemical species and (ii) a solvent,such that the microdroplet impinges a second chemical species and formsa third chemical species therewith, the solvent having a boiling pointof 150° C. or above, a surface tension of 30 dynes/cm or above, and aviscosity of 0.015 g/(cm)(sec) or above.

[0022] The invention also provides a fully automated solution forsynthesizing oligonucleotides, particularly deoxyribonucleosides andribonucleosides, by repeatedly cycling a substrate through steps ofdepositing nucleoside monomers and of treating the substrate by rinsingoff unattached nucleoside monomers. A system in accordance with theinvention includes an inkjet print head for spraying nucleoside monomerson a substrate, a scanning transport for moving the substrate withrespect to the print head so that the monomer is deposited at specifiedsites, a flow cell for treating the substrate deposited with the monomerby exposing the substrate to selected fluids, a treating transport formoving the substrate between the print head and the flow cell fortreatment in the flow cell, and an alignment unit for aligning thesubstrate so that the substrate is correctly positioned with respect tothe print head each time the substrate is positioned for deposition.Computer-controlled motion stages and vacuum chucks are used to move thesubstrate during deposition and to move the substrate between the printhead and the flow cell.

[0023] Each time the substrate is picked up by a vacuum chuck and placedover the print head, the substrate is positionally calibrated by using acamera in conjunction with marks that are placed on the substrate thefirst time it is handled. Translational misalignment is corrected bymoving the vacuum chuck in two axes of linear motion. Rotationalmisalignment is corrected by physically rotating the vacuum chuck withina substrate holder.

[0024] Software, programmed apparatuses, and computer readable memory,for carrying out the methods of the invention are also provided.

[0025] The present invention may be understood more fully by referenceto the following figures, detailed description and illustrative exampleswhich are intended to exemplify non-limiting embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1a is a copy of a photograph of water condensed onto an arrayof approximately 250 surface tension wells. Individual droplets areconfined to square regions of 100 micron sides by 30 micron widehydrophobic barriers.

[0027]FIG. 1b is a side view of a surface tension well showing thearrangement of hydrophilic and hydrophobic regions, and a cross sectionof a reagent drop sitting on an hydroxylated hydrophilic surface. Thebottom layer substrate is silicon dioxide, and repeating units of —Frepresent a perfluorinated hydrophobic surface. The reagent drop sits onrepeating —OH units of the silicon dioxide support. The diameter of thereagent drop is approximately 100 μm.

[0028]FIG. 2 is a schematic diagram of a piezoelectric pump in an inkjetprint head.

[0029]FIG. 3 shows the substrate with surface tension wells, moved by anX-Y translation stage, above the nozzles spraying microdroplets.

[0030]FIG. 4 is a scheme showing a complete cycle of oligonucleotidesynthesis comprising (a) delivering a reactant to each well, (b) washingaway unreacted monomers, and (c) deprotecting the ends of the extendedmolecules.

[0031]FIG. 5 shows an automated system for synthesizing oligonucleotidesin accordance with the invention.

[0032]FIG. 6 shows inkjet print heads used in the system of FIG. 5.

[0033]FIG. 7 shows a scanning transport used in the system of FIG. 5.

[0034]FIG. 8 shows an alignment unit used in the system of FIG. 5.

[0035]FIG. 9 shows a flow cell used in the system of FIG. 5.

[0036]FIG. 10 shows a transfer station used in the system of FIG. 5.

[0037]FIG. 11 is a block diagram showing a computer and controlcomponents used in conjunction with the system of FIG. 5.

[0038]FIG. 12 is a block diagram of a controller used to control theinkjet print heads.

[0039]FIG. 13 is a flow chart depicting the operation of the computersoftware used to initialize the inkjet print heads.

[0040]FIG. 14 is a flow chart depicting the operation of the softwareused to control the operation of the automated synthesis system.

[0041]FIG. 15 is a flow chart depicting the operation of the softwareused to control the operation of the scanning transport and theoperation of the flow cell.

[0042]FIG. 16 is a flow chart depicting the operation of the softwareused to align a substrate relative to the print heads.

[0043]FIG. 17 is a flow chart depicting the operation of the softwareused to further align the substrate.

[0044]FIG. 18 is a flow chart depicting the operation of the softwareused to further control the operation of the flow cell.

[0045]FIG. 19 is a flow chart depicting the operation of the softwareused to measure the center positions of registration marks used foralignment.

[0046]FIG. 20 is a flow chart depicting the operation of the softwareused to calculate the slope and equation of a line detected by a cameraduring alignment.

[0047]FIG. 21 is a flow chart depicting the operation of the softwareused control the deposition of a layer of nucleoside monomers.

DETAILED DESCRIPTION OF CHEMICAL SYNTHESIS USING MICRODROPLETSMICRODROPLETS

[0048] The present invention relates to a microdroplet of a solution,the solution comprising a high surface tension solvent having a boilingpoint of about 150° C. or above, a surface tension of about 30 dynes/cmor above, and a viscosity of about 0.015 g/(cm)(sec) or above. Eachmicrodroplet is a separate and discrete unit, preferably having a volumeof about 100 pL or less, more preferably about 50 pL or less. Suchmicrodroplets are useful for synthesis of chemical compounds, and inparticular, for the synthesis of arrays of chemical compounds that arearranged in microdots which are separate and discrete units. It will beunderstood by those skilled in the art that a “solution” comprises“solvent” and “solute”. In the present instance, the “solute” ispreferably a chemical species that is a reagent, as described below. Asused herein, “microdot” refers to a microdroplet that is associated witha substrate.

[0049] The arrays of chemical compounds synthesized by the methods ofthe invention are useful as libraries of chemical probes. Where thedifferent chemical compounds obtained by the methods of the presentinvention are peptides, the peptide arrays can be contacted with aprotein or peptide of known sequence, such as an antibody, a cellreceptor or other type of receptor, so as to identify a peptide,synthesized according to the present invention, that is capable ofbinding to the peptide of known sequence. Such a peptide can be readilysequenced by methods well known to those skilled in the art. Where thedifferent chemical compounds synthesized by the present invention areoligonucleotides, the oligonucleotide arrays can be used ashybridization probes, for example, for genotyping or expressionanalysis, e.g., as a tool in gene therapy whereby mutations may beidentified in a genome, or to identify DNA in samples from theenvironment, or may be used to synthesize complementary oligonucleotidesby using DNA polymerase and primers, or as primers for DNA sequencing orpolymerase chain reaction. Where the different chemical compoundssynthesized by the present invention are peptides, oligonucleotides, orother chemical species such as polysaccharides or other biologicallyactive molecules, such chemical species can be subjected to a variety ofdrug screening assays to identify and ascertain their efficacy.

[0050] As stated above, the microdroplets of the present invention arein the form of separate and discrete units. By this is meant that themicrodroplets that comprise the first chemical species do not intermixprior to impinging those microdots that comprise the second chemicalspecies. As also stated above, the arrays of compounds that are obtainedin accordance with the present invention are arranged in microdots whichare separate and discrete units. By this is meant that each microdropletthat impinges a second chemical species is delivered such that theresulting microdots which each comprise a third chemical species do notoverlap or intermingle. It is to be pointed out, however, that thesecond chemical species need not be arranged in separate and discreteunits prior to reaction with the first chemical species; for example, asubstrate having a plurality of functional groups that are not set apartfrom each other in separate domains can be impinged by microdroplets atseparate and discrete loci, resulting in the formation of separate anddiscrete microdots of a third chemical species which are separated fromeach other via the unreacted second chemical species.

SOLVENTS FOR MICRODROPLETS

[0051] The present inventor has found, surprisingly and unexpectedly,that high surface tension solvents that have a boiling point of 150° C.or above, a surface tension of 30 dynes/cm or above, and a viscosity of0.015 g/(cm)(sec) or above, give rise to microdroplets that haveproperties that are optimal for microdroplet formation and stability,particularly when used as a reaction solvent for the synthesis of arraysof organic compounds such as oligonucleotides and peptides. It is to bepointed out that the boiling point, surface tension and viscosity valuesof the present solvents are those obtained when measured at or around760 mm/Hg, and at or around room temperature (approximately 22° C.).

[0052] For example, the present microdroplets, which comprise solventsthat have boiling points of 150° C. or above, or thereabout, overcomethe disadvantages of those microdroplets that comprise lower boilingsolvents by not readily evaporating upon formation or deposition. Thischaracteristic is especially important when the microdroplets are to beused as vehicles for chemical reagents: where a reactive chemicalspecies contained in one microdroplet seeks to react with a reactivechemical species contained in a second microdroplet that is impinged bythe first, the use of relatively high boiling solvent, as in presentmicrodroplets, ensures that the solvent does not evaporate prior toreaction between the two reactive chemical species. In addition,microdroplets that are formed from solvents that have boiling points of150° C. or above do not appreciably evaporate upon formation, whichprevents (a) deposition of microdroplet solutes around or within themicrodroplet generating source, and accordingly prevents clogging; and(b) unwanted precipitation of solutes onto the array surface.

[0053] It has also been found that microdroplets, particularly thosehaving a volume of about 100 pL or less, that comprise solvents thathave surface tensions of 30 dynes/cm or above, or thereabout, have arelatively low affinity for the face of a nozzle used to generatemicrodroplets and accordingly, are more stable and uniformly sized.These properties are particularly desirable when the amount of solute,e.g., a reactive chemical species, that is to be dispensed as amicrodroplet solution, should preferably be uniform from microdroplet tomicrodroplet, such as for example in the case of organic synthesis. Inaddition, microdroplets that have a relatively low affinity for the faceof a nozzle can be dispensed more efficiently than those that have arelatively high affinity for the face of a nozzle.

[0054] It has further been found that microdroplets, particularly thosehaving a volume of about 100 pL or less, that comprise solvents thathave viscosity values of 0.015 g/(cm)(sec) or above, or thereabout, donot succumb to residual oscillations caused by the microdropletgenerating device and accordingly, maintain their structural integrity,e.g., spherical shape, when dispensed. This property is particularlyimportant when the dispensed microdroplets are to be deposited inclosely packed arrays of uniformly shaped microdots that cannot overlap.

[0055] In addition, solvents that have the above boiling point, surfacetension and viscosity properties do not appreciably initiate thedegradation or decomposition of synthetic polymers that are commonlyused in microdroplet dispensing devices, allowing them to be used inconjunction with a variety of plastic parts or components.

[0056] Furthermore, where the solvent is to be used for organicsynthesis, the solvent molecules must not comprise, or must not bemodified so as to comprise, reactive functional groups, such ashydroxyl, primary amino, secondary amino, sulfhydryl, carboxyl, andanhydride groups, that can easily interfere, i.e., react, with astarting material, reagent, intermediate or product chemical species.

[0057] The present inventor has found that a class of organic solventsthat (a) has (i) a boiling point of 150° C. or above, (ii) a surfacetension of 30 dynes/cm or above, and (iii) a viscosity of 0.015g/(cm)(sec); and (b) is represented by the formula (I):

[0058] wherein

[0059] A=O or S;

[0060] X=O, S or N(C₁-C₄ alkyl);

[0061] Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

[0062] R=C₁-C₂₀ straight or branched chain alkyl, is particularlypreferred for use in chemical synthesis where a first chemical speciesis delivered to a second chemical species in the form of a microdroplet.

[0063] As used herein, “branched chain alkyl” refers to a C₁-C₁₉straight chain alkyl group substituted with one or more methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,(1-methyl)butyl, (2-methyl)butyl, (3-methyl)butyl and neopentyl groups,or the like; wherein the total number of carbon atoms of the branchedchain alkyl does not exceed twenty.

[0064] Preferably, —R— is a branched chain alkyl, and has the formula—CH(CH₃)—, —CH₂CH₂—, —CH(CH(CH₃)₂)—, —CH(CH(CH₃))CH— or —CH₂CH(CH₃)—.Especially preferred solvents of formula (I) include, but are notlimited to:

[0065] N-methyl-2-pyrrolidone (boiling point=202° C.; surfacetension=40.7 dynes/cm; and viscosity=0.017 g/(cm)(sec));

[0066] 2-pyrrolidone (boiling point=245° C.; surface tension=46.9dynes/cm; and viscosity=0.13 g/(cm)(sec));

[0067] propylene carbonate (boiling point=240° C.; surface tension=40.7dynes/cm; and viscosity=0.025 g/(cm)(sec));

[0068] γ-valerolactone (boiling point=208° C.; surface tension=30.9dynes/cm (at 51° C.); and viscosity=0.033 g/(cm)(sec));

[0069] 6-caprolactam (boiling point=270° C.; surface tension=42 dynes/cm(at 69° C.); and viscosity=0.12 g/(cm)(sec) (at 70° C.));

[0070] ethylene carbonate (boiling point=248° C.; surface tension=42.6dynes/cm (at 37° C.); and viscosity= 0.012 g/(cm)(sec) (at 38° C.));

[0071] γ-butyrolactone (boiling point=206° C.; surface tension=36.5dynes/cm (at 43° C.); and viscosity=0.017 g/(cm) (sec));

[0072] δ-valerolactone (boiling point=218-220° C.; surface tension andviscosity values not available);

[0073] 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (boilingpoint=230° C. (754 mm/Hg); surface tension=36.12 dynes/cm; andviscosity=0.029 g/(cm)(sec));

[0074] ethylene trithiocarbonate (boiling point= 307° C.; surfacetension and viscosity values not available); and

[0075] 1,3-dimethyl-2-imidazolidinone (boiling point= 220° C. (754mm/Hg; surface tension=37.6 dynes/cm; and viscosity=0.019 g/(cm)(sec)).

[0076] Propylene carbonate solvent is most preferred.

[0077] It is to be pointed out that boiling point values increase as thepressure increases, and surface tension and viscosity values increase asthe temperature decreases. Accordingly, the boiling point values arehigher at 760 mm/Hg than at certain lower pressures reported above, andthe surface tension and viscosity values are higher at room temperaturethan at certain higher temperatures reported above.

[0078] It is also to be pointed out that the solvents of the inventiondo not necessarily have to exhibit all three characteristics of having aboiling point of about 150° C. or above, a surface tension of about 30dynes/cm or above, and a viscosity of about 0.015 g/(cm)(sec) or aboveto be useful in the methods, apparatus or automated system of theinvention. For example, solvents which exhibit less than the valuesdescribed above for one or more of the three physical properties canalso be used so long as the solvents maintain their ability to supportbiopolymer synthesis and are capable of forming discrete microdropletswithout substantially initiating degradation of components of theapparatus or automated system. Such solvents can exhibit, for example,uncharacteristically high values for one or more of three physicalproperties which can be compensated by a corresponding decrease in thevalue of another one of the above physical properties. Moreover,solvents that have values less than those described above for eitherboiling point, surface tension or viscosity can similarly be compensatedby, for example, substituting or modifying the components of theapparatus so as to maintain the ability of the ink jet head, forexample, to dispense discrete microdroplets of solvent. Thus, thesolvents of the invention can exhibit values for one or more physicalproperties less than those described above so long as they maintaintheir function of supporting synthesis in microdroplets. Given theteachings herein, those skilled in the art will know or can determinewhich solvents can be used in the methods of the invention.

PREPARATION OF MICRODROPLETS

[0079] The microdroplets of the present invention are preferablyobtained by forcing the solvent, at a rate of about 1 to about 10 m/sec,through an orifice or nozzle that has a diameter of about 10 to about100 μm. It is critical that the microdroplets so obtained are dispensedfrom the orifice or nozzle in the form of separate and discrete units.

[0080] One embodiment of the invention involves a system utilizing amechanism for localizing and separating microdroplets preferably havinga volume of about 100 pL or less, more preferably about 50 pL or less.The microdroplets are separated from each other, in the form ofmicrodots by, for example, hydrophobic domains. At such small solventvolumes, surface tension is the strongest force that acts on amicrodroplet, and can be used, for example, to create circular “surfacetension wells” (FIG. 1a and FIG. 1b), preferably arranged in arrays ofmicrodots. Such surface tension wells can constrain each microdot, andprevent adjacent microdots from overlapping or merging with each other.According to the invention, methods have been developed that produce anarray of microdots that are in the form of circular wells. The microdotsdefine the locations of the array elements, and act as miniaturereaction vessels for chemical synthesis. The microdots can vary in sizeand will depend on the intended use of the synthesized array. Forexample, the diameter of each microdot can be greater than 1000 μm, buttypically ranges from about 1 to about 1000 μm, preferably from about 10to about 500 μm, and more preferably from about 40 to about 100 μm.Similarly, the distance between adjacent microdots will vary accordingto the intended use of the array. The distance between each microdot istypically from about 1 to about 500 μm, preferably from about 10 toabout 100 μm, and more preferably from about 20 to about 30 μm. Thoseskilled in the art will know or can determine without undueexperimentation what is the appropriate separation of microdots withinan array for a particular use.

[0081] Physical separation of circular wells can be accomplishedaccording to known methods. For example, such methods can involve thecreation of hydrophilic wells by first applying a protectant, or resist,over selected areas over the surface of a substrate. The unprotectedareas are then coated with a hydrophobic agent to yield an unreactivesurface. For example, a hydrophobic coating can be created by chemicalvapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane ontothe exposed oxide surrounding the protected circles. Finally, theprotectant, or resist, is removed exposing the well regions of the arrayfor further modification and nucleoside synthesis using the high surfacetension solvents described herein and procedures known in the art suchas those described by Maskos & Southern, Nucl. Acids Res. 20:1679-1684(1992). Alternatively, the entire surface of a glass plate substrate canbe coated with hydrophobic material, such as3-(1,1-dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablatedat desired loci to expose the underlying silicon dioxide glass. Thesubstrate is then coated with glycidyloxypropyl trimethoxysilane, whichreacts only with the glass, and which is subsequently “treated” withhexaethylene glycol and sulfuric acid to form an hydroxyl group-bearinglinker upon which chemical species can be synthesized (U.S. Pat. No.5,474,796 to Brennan). Arrays produced in such a manner can localizesmall volumes of solvent within the circular wells by virtue of surfacetension effects (L'opez et al., Science 260:647-649 (1993)).

[0082] The protectant, or resist, can be applied in an appropriatepattern by, for example, a printing process using a rubber stamp, asilk-screening process, an inkjet printer, a laser printer with asoluble toner, evaporation or by a photolithographic process, such asthat reported by Kleinfeld, D., J. Neurosci. 8:4098-4120 (1988). Thehydrophobic coating can also be applied directly in any appropriatepattern by, for example, a printing process using a rubber stamp, asilk-screening process, or laser printer with a hydrophobic toner.

[0083] Additionally, the use of the present solvents allows for thedirect synthesis of chemical compound arrays onto a substrate such as asilicon wafer or a glass slide without the need for creating hydrophilicwells. Such direct synthesis is accomplished, for example, by accuratelydepositing a microdroplet, of a solution comprising a first chemicalspecies, at each loci of the array. As described above, inkjet printheads can be used for accurately dispensing microdroplets in eithersingle or multiple dispenser format, i.e., from either a single nozzleor from multiple nozzles, or with the dispensation of either a singlemicrodroplet or of multiple microdroplets.

[0084] The present invention also encompasses a method for delivering afirst chemical species to an appropriate locus of the substrate. In oneembodiment, microfabricated piezoelectric pumps, or nozzles, similar tothose used in inkjet printers, are used to deliver a specified volume ofsolution to an appropriate locus of the substrate (Kyser et al., J.Appl. Photographic Eng., 7:73-79 (1981)).

[0085]FIG. 2 shows an example of a piezoelectric pump, described by wayof example but not limitation as follows: The piezoelectric pump is madeby using etching techniques known to those skilled in the art tofabricate a shallow cavity in silicon base 1. A thin, glass membrane 3is then anodically bonded to silicon base 1 to seal the etched cavity,thus forming a small cavity 2 with narrow inlet 5 and nozzle 7. When theend of inlet 5 of the piezoelectric pump is dipped in the reagentsolution, capillary action draws the liquid into the cavity 2 until itcomes to the end of the nozzle 7. When an electrical pulse is applied tothe piezoelectric element 4 glued to the glass membrane it bows inward,ejecting a microdroplet 6 out of the nozzle at the end of thepiezoelectric pump. The cavity refills itself through inlet 5 bycapillary action. Simple designs for piezoelectric pumps will operate at1 thousand cycles per second (kilo Hertz or kHz), while more advanceddesigns operate at 6 kHz (See Takahashi et al., NEC Res. and Develop.80:38-41 (1986)).

[0086] For chemical synthesis in two dimensional arrays, piezoelectricpumps that will deliver on demand microdroplets having a volume of about100 pL of less, at rates of several hundred Hz, are preferred. However,the microdroplet volume or speed at which the piezoelectric pump canoperate may vary depending on the need. For example, if an array havinga greater number of microdots but with the same array surface area is tobe synthesized, then smaller microdroplets should be dispensed.Additionally, if synthesis time is to be decreased, then the operationspeed of the microdroplet dispensing device can be increased. Adjustingsuch parameters is within the purview of one skilled in the art, and canbe performed according to the need.

[0087]FIG. 3 shows substrate 8 being “scanned” (moved) across a set ofnozzles 9 using a computer-controlled X-Y translation stage whichtranslocates the nozzles relative to the substrate, or preferably,translocates the substrate relative to the nozzles. The computersynchronizes and times the firing of the nozzles 9 to deliver a singlemicrodroplet 10 of the appropriate first chemical species 11 to eachlocus of the substrate.

[0088]FIG. 4 illustrates a cycle to synthesize an oligonucleotide. Itbegins by delivering a solution comprising an appropriatelyfunctionalized nucleoside either along with a catalyst such as5-ethylthiotetrazole premixed with the nucleoside, or separately, from aseparate nozzle, to each well on the substrate. The entire substrate canthen be rinsed to remove excess monomer; exposed to an oxidizingsolution, typically an iodine/tetrahydrofuran/pyridine/water mixture;and then rinsed with acid to deprotect the 5′ end of the oligonucleotidein preparation for the next round of synthesis. The rinses can be commonto all the microdots of the substrate and can be performed, for example,by bulk immersion of the substrate. One such iteration adds a firstchemical species to each growing oligomer; thus, an array of oligomershaving a length of ten units each requires 10 such iterations.

[0089] The number of iterations, and therefore, the length of theoligomers obtained, will be determined by the need and desired use forthe array. As such, the oligomer lengths which can be achieved using themethods of the invention are limited only by existing couplingchemistries. Routinely, oligomers having about 10 to about 100, andpreferably having about 20 to about 60 units each can be synthesized. Asnew coupling chemistries emerge, so will the yield and length ofoligomeric products. Therefore, it is envisioned that the methods of theinvention are useful for the synthesis of oligomer arrays of greaterthan 100 units each.

[0090] Inkjet printers generally contain print heads having 50 to 100independently controlled nozzles. With each nozzle operating at severalhundred Hz, an apparatus with five such heads can deliver a microdropletof a solvent comprising a first chemical species to 100,000 differentloci in a matter of seconds. A complete synthesis cycle can take, forexample, 5 minutes, or just over 2 hours for an array of 100,000oligomers having 25 units each. Inkjet print heads having a greater orfewer number of nozzles, and which operate at different speeds, can beused as well. Additionally, multiple heads can be simultaneously used tosynthesize the arrays. Such modifications are known to those skilled inthe art and will vary depending on the size, format and intended use ofthe assay.

CHEMICAL SYNTHESIS USING MICRODROPLETS

[0091] The microdroplets of the present invention further comprise afirst chemical species which is soluble in a solvent of the inventiondescribed in Section 5.2. Typically, upon formation, the microdroplet isa solution of the first chemical species having a concentration of about1 nM to about 5M, preferably from about 0.01 mM to about 1M. Themicrodroplet impinges a second chemical species, and the first chemicalspecies of the microdroplet reacts with the second chemical species toform a third chemical species, the third chemical species beingdifferent from the first and second chemical species. In this manner,and particularly when the second chemical species is linked to asubstrate, arrays of different chemical compounds, arranged in microdotswhich are separate and discrete units, can be synthesized.

[0092] The first chemical species is any chemical compound that canreact with a second chemical species so as to form a third chemicalspecies. The first chemical species and the second chemical species canbe the same or different, but the third chemical species must bedifferent from the first chemical species and the second chemicalspecies. The process of reacting a first chemical species with a secondchemical species to form a third chemical species may be repeated at thesite of the third chemical species, such that in a subsequent iterationof the process, the third chemical species becomes the “second chemicalspecies” with respect to an impinging microdroplet comprising a firstchemical species, and the reaction product of that “second chemicalspecies” and the first chemical species is a new third chemical speciesthat is different from the original third chemical species. Accordingly,as used herein, “second chemical species” is that which reacts with afirst chemical species, and “third chemical species” is the reactionproduct of the first chemical species and second chemical species. Anunlimited number of iterations of this process can be performed untilthe desired chemical compound is synthesized. In a specific embodiment,the third chemical species is an oligomer (e.g., a homo-oligomer orhetero-oligomer), preferably a biopolymer, containing as monomer unitsthe first and second chemical species.

[0093] In one embodiment, the first chemical species reacts with thesecond chemical species in the presence of a catalyst. Accordingly, thesolution can optionally comprise a catalyst, such as an enzyme or otherchemical catalyst, that accelerates the rate of reaction between thefirst chemical species and second chemical species. Alternatively, if itis advantageous that the first chemical species react with the secondchemical species in the presence of a catalyst, a solution comprising acatalyst can be delivered to the locus where the first chemical speciesimpinges the second chemical species either prior or subsequent to theimpingement of the second chemical species by the first chemicalspecies.

[0094] For ease of handling, the second chemical species can beassociated with a substrate. By “associated with” is meant (a) adheres,but is not chemically attached, to, such as for example where the secondchemical species is in the form of a microdot on a substrate of paper oruntreated glass, or in solution sitting in a microwell or microcavity ofthe substrate; or (b) is chemically attached to, such as for examplewhere the second chemical species is covalently bonded directly to afunctional group of the substrate, or bonded to a linker that isattached to the substrate.

[0095] As used herein, the term “substrate” is intended to mean agenerally flat surface, porous or not, which has, or can be chemicallymodified to have, reactive groups suitable for attaching further organicmolecules. Examples of such substrates include, but are not limited to,glass, silica, silicon, polypropylene, TEFLON®, polyethylimine, nylon,fiberglass, paper, and polystyrene. Bead structures may also be attachedto the surface of the substrate, wherein the beads are composed of oneor more of the preceding substrate materials. As used herein, substrateswhich contain or are modified to contain chemically reactive species cantherefore also be referred to as a “chemical species.”

[0096] Where the third chemical species is to be assayed, for example,for biological activity, it is preferable that the third chemicalspecies be readily removable from the substrate: e.g., in the case wherethe third chemical species adheres, but is not chemically attached, to,the substrate, by washing with a suitable solvent; in the case where thethird chemical species is in solution sitting in a microwell ormicrocavity of the substrate, by removing the solution via amicropipetting or microsyringing device; and in the case where the thirdchemical species is chemically attached to the substrate (eitherdirectly or via a linker), by releasing, preferably hydrolyzing orenzymatically cleaving, the third chemical species from the substrate orlinker attached to the substrate. It will be understood that in thelatter instance, the third chemical species so released will be slightlychemically modified relative to the attached third chemical species; forexample, where the third chemical species is attached to an hydroxyl oramino group of the substrate via an ester or amide bond, the thirdchemical species so hydrolyzed will have a terminal carboxyl orcarboxylate group. Accordingly, the term “third chemical species” isalso meant to encompass the chemical species that is ultimately releasedfrom the substrate.

[0097] In one embodiment, the first chemical species is, for example, anucleoside, activated nucleoside, or nucleotide; the second chemicalspecies is, for example, a substrate having reactive functional groups,a linker attached to a substrate, or a nucleoside, nucleotide, oroligonucleotide attached to either the linker or directly to thesubstrate; and the third chemical species is a nucleoside activatednucleoside, or nucleotide (in the case where the second chemical speciesis a substrate or linker attached to a substrate) or an oligonucleotideof at least two nucleoside units (in the case where the second chemicalspecies is a nucleoside or oligonucleotide), chemically attached toeither the linker or directly to the substrate.

[0098] Preferably, the first chemical species is a nucleoside having anactivated phosphorous-containing, preferably a phosphoramidite, group atthe 3′ position, and a protected hydroxyl group at the 5′ position, andthe second chemical species is (a) a substrate or linker attached to asubstrate having an thiol or hydroxyl group that is capable of forming astable, covalent bond with the phosphoramidite group at the 3′ positionof the first chemical species, or (b) a nucleoside or oligonucleotideattached to either the linker or directly to the substrate, and havingan thiol or hydroxyl group at its 5′ position that is capable of forminga stable, covalent bond with the phosphoramidite group at the 3′position of the first chemical species.

[0099] Thus, where the first chemical species is a nucleoside, thepresent invention encompasses a solution, preferably in microdropletform, comprising a solvent and a nucleoside, wherein the solvent has aboiling point of 150° C. or above, a surface tension of 30 dynes/cm orabove, and a viscosity of 0.015 g/(cm)(sec) or above. Preferably, thesolvent is represented by the formula (I), described in Section 5.2,above.

[0100] As used herein, the term “nucleoside” encompasses bothdeoxyribonucleosides and ribonucleosides, and the term “oligonucleotide”refers to an oligonucleotide that comprises deoxyribonucleotide orribonucleotide units, such that the term “oligonucleotide” encompassesboth oligodeoxyribonucleotides and oligoribonucleotides.

[0101] Where the first chemical species and second chemical species bearadditional reactive groups, such as for example primary amino groups ofadenine, cytosine and guanine bases, those reactive groups can haveadditional protecting groups, so as to preclude unwanted side reactionsif not protected. The primary amino groups of adenine, cytosine andguanine bases are protected with amino protecting groups well known tothose skilled in the art, preferably, with t-butylphenoxyacetyl (tBPA)groups.

[0102] Typically, by way of example, a solution comprising a nucleosideas the first chemical species and having (a) a protecting group,preferably a monomethoxytrityl or dimethoxytrityl protecting group, onthe 5′ hydroxyl group, (b) an activated phosphorous-containing group atthe 3′ position, and (c) another protecting group, preferably a tBPAgroup, at any primary amino group of the base portion of the nucleoside,in a solvent of the invention is dispensed as a microdroplet onto asecond chemical species, e.g., a substrate or a substrate with a linkerhaving, for example, hydroxyl functional groups.

[0103] Suitable nucleosides useful for the synthesis of oligonucleotidesaccording to the present methods are those nucleosides that containactivated phosphorous-containing groups such as phosphodiester,phosphotriester, phosphate triester, H-phosphonate and phosphoramiditegroups. It will be understood that where the first chemical species is anucleoside, and the second chemical species is a nucleoside or anoligonucleotide, the first and second chemical species have the sameactivated phosphorous-containing group. Such activated nucleosides andtheir relevant chemistries are described in, for example, Nucleic Acidsin Chemistry and Biology (Blackburn and Gait eds., 2d ed. 1996) and T.Atkinson et al., Solid-Phase Synthesis of Oligodeoxyribonucleotides bythe Phosphite-Triester Method, in Oligonucleotide Synthesis 35-39 (M. J.Gait ed., 1984). Preferably, the activated phosphorous-containing groupis a phosphoramidite, more preferably a phosphoramidite having acyanoethyl group, and most preferably, a phosphoramidite having theformula (iPr₂N)P(OCH₂CH₂CN)OR, where R is the 3′ position of anucleoside. By way of example but not limitation, a detailed example ofoligonucleotide synthesis using a phosphoramidite nucleoside derivativeis described below.

[0104] The reaction between the 3′ phosphoramidite group of thenucleoside, and the hydroxyl groups of the substrate-bound linker, isfacilitated by a catalyst, such as 5-methylthiotetrazole, tetrazole, orpreferably 5-ethylthiotetrazole. The solution of nucleoside canadditionally comprise the catalyst or preferably, following dispensationof the nucleoside solution, an additional microdroplet of catalystsolution can be dispensed upon the locus at which the nucleosidesolution impinged the substrate-bound linker. The reaction between thehydroxyl groups of the substrate-bound linker, and the 3′phosphoramidite group of the nucleoside, preferably performed in thepresence of the catalyst, forms a protected nucleoside anchored to thesubstrate via a 3′ phosphite group. This protected nucleoside is now thethird chemical species. Preferably, the entire substrate is washed witha solvent, e.g., acetonitrile or dichloromethane, before proceeding tothe next step. It will be appreciated that a phosphoramidite group willform a phosphite group, preferably in the presence of a catalyst, with ahydroxyl group in general. Such an hydroxyl group may be either aprimary, secondary or tertiary alcohol, or may be of a silanol. Thephosphite group is oxidized to a phosphate group in the presence of anoxidizing agent. Additionally, a phosphoramidite group will form athiophosphite group, preferably in the presence of a catalyst, with athiol group in general. Such a thiol group may be either a primary,secondary or tertiary thiol. The thiophosphite group can be oxidized toa thiophosphate group in the presence of an oxidizing agent.

[0105] The resulting 3′ phosphite group is then oxidized to a 3′phosphate group. Preferably, the oxidizing agent used to oxidize thephosphite group to the phosphate group is iodine, more preferably, asolution of iodine, water, an organic base such as pyridine, and anorganic solvent such as tetrahydrofuran. Preferably, the entiresubstrate, to which the nucleoside having the 3′ phosphite group isattached, is washed with the oxidizing agent, oxidizing the 3′ phosphitegroup to the 3′ phosphate group. In one embodiment of the invention, thesubstrate, to which the nucleoside having the 3′ phosphite group isattached, is submerged in a bath containing the oxidizing agent.Alternatively, the oxidizing agent can be dispensed as a microdropletonto the locus at which the nucleoside, having the 3′ phosphite group,is synthesized. In such an instance, the oxidizing agent is preferablydispensed as a solution in a solvent described in Section 5.2. Followingtreatment with the oxidizing agent, and before proceeding to the nextstep, the entire substrate is preferably washed with a solvent, e.g.,acetonitrile or dichloromethane.

[0106] Following oxidation, the entire substrate, to which thenucleoside having the 3′ phosphate group is attached, is treated with areagent that “caps” the unreacted hydroxyl groups of the substrate-boundlinker so as to prevent them from competing for the phosphoramiditegroup of a subsequently dispensed nucleoside with the 5′ position of thenewly added nucleoside, above. Preferably, the capping reagent is anacylating agent, more preferably, an acyl halide and most preferably,perfluorooctanoyl chloride. Preferably, the entire substrate is washedwith a solvent, e.g., acetonitrile or dichloromethane, before proceedingto the next step.

[0107] In the next step, the nucleoside having the 3′ phosphate groupthat is covalently bonded to the linker of the substrate is treated witha first deprotecting agent which removes the protecting group from thebound nucleoside's 5′ position, exposing a reactive hydroxyl group atthe 5′ position. Preferably, the first deprotecting agent is an acid,and more preferably dichloroacetic acid. In a preferred embodiment, theentire substrate to which the nucleoside having the 3′ phosphate groupis bonded is rinsed with a solution of the first deprotecting agent.Alternatively, the first deprotecting agent can be dispensed as amicrodroplet; in such a case, the microdroplet preferably comprises asolvent described in Section 5.2. Before proceeding to the next step,the entire substrate is preferably washed with a solvent, e.g.,acetonitrile or dichloromethane.

[0108] In the following step, a second nucleoside having an activatedphosphorous-containing, preferably a phosphoramidite, group at the 3′position, and a protected hydroxyl group at the 5′ position, isdispensed as a microdroplet solution using a solvent described inSection 5.2 so as to impinge the microdot.

[0109] The solution of nucleoside can additionally comprise a catalyst,or alternatively, in a subsequent step, a microdroplet of a solution ofcatalyst, such as 5-methylthiotetrazole, tetrazole, or preferably5-ethylthiotetrazole, preferably in a solvent described in Section 5.2,is dispensed upon the locus at which the second nucleoside solutionimpinged the microdot. The catalyst facilitates a reaction between the5′ hydroxyl group of the first nucleoside and the 3′ phosphoramiditegroup of the second nucleoside, resulting in the coupling of the secondnucleoside to the first nucleoside via a phosphite group, as describedabove.

[0110] At this point, successive iterations of (a) oxidizing theresulting phosphite to a phosphate group; (b) removing the 5′ protectinggroup; (c) dispensing an additional protected nucleoside having aphosphoramidite group at its 5′ position, optionally in the presence ofcatalyst or, preferably; (d) dispensing the catalyst at the locus wherethe additional nucleoside was dispensed, preferably with solvent washingsubsequent to performing each of iterative steps (a)-(d), affords alinker-bound oligonucleotide that has a 2-cyanoethylphosphate group, aswell as a protecting group, preferably a tBPA protecting group, on anyprimary amino group of the nucleoside bases.

[0111] Treatment with a second deprotecting agent, preferablyethanolamine, removes the protecting groups from the nucleoside bases,and converts the oligonucleotide 2-cyanoethylphosphate groups tophosphate groups. Preferably, the entire substrate to which theoligonucleotide is bonded is rinsed with a solution of the seconddeprotecting agent. Alternatively, the second deprotecting agent can bedispensed as a microdroplet; in such a case, the microdroplet preferablycomprises a solvent described in Section 5.2.

[0112] The chemistry relating to the above-described example ofoligonucleotide synthesis is summarized below in Scheme 1:

[0113] It is to be pointed out that this process may be repeated atdifferent loci of the substrate, using different first chemical speciesand second chemical species, so as to obtain, if desired, a differentoligonucleotide at each loci.

[0114] The oligonucleotides thus synthesized can be used in theirsubstrate-anchored form, e.g., in hybridization assays conducted on thesubstrate. Alternatively, in another embodiment of the invention, thesubstrate-anchored oligonucleotides obtained above can be cleaved fromthe substrate. In a specific embodiment, the nucleoside unit of theoligonucleotide that is directly attached to the substrate, or, attachedto a linker that is attached to the substrate, is attached to thesubstrate, or to the linker, via an ester bond. Such an ester bond issusceptible to hydrolysis via exposure to a hydrolyzing agent. Such anester bond is preferably formed on the first nucleoside prior toapplication to the substrate. In this embodiment, prior to synthesis ofthe oligonucleotide to be cleaved, an amino group, preferably in theform of a long chain alkylamine, is attached to the substrate (see T.Atkinson et al., Solid Phase Synthesis of Oligodeoxyribonucleotides bythe Phosphitetriester Method, in Oligonucleotide Synthesis (M. J. Gaited., 1984). The first nucleoside having the ester bond is attached tothe amino group of the substrate via an activated O-succinate group(Scheme 2, below, and T. Atkinson et al., Solid-Phase Synthesis ofOligodeoxyribonucleotides by the Phosphitetriester Method, inOligonucleotide Synthesis (M. J. Gait ed., 1984), which reacts with theamino group of the substrate to form an amide bond therewith. As usedherein, “activated” O-succinate groups are those that have, at thesuccinate carbonyl group not attached to the nucleoside, a leaving groupthat is capable of being displaced by an amino group, preferably anamino group of a substrate. Preferably, the activated O-succinate groupis one that has, at the succinate carbonyl group not attached to thenucleoside, a p-nitrophenoxy group. Methods for preparing activatedO-succinate groups are well known to those skilled in the art. Such anactivated nucleoside can be applied to the substrate as a microdropletsolution.

[0115] It should be noted that in the instance where a polymercontaining both nucleoside and non-nucleoside monomer units is desiredto be synthesized, a second or subsequent chemical species to be bondedto the first nucleoside can be any phosphoramidite-containing compound,such as for example, phosphoramidite-modified amines, thiols,disulfides, ethylene glycols and cholesterol derivatives. Suchphosphoramidite-containing compounds are commercially available fromGlen Research, Sterling, Va.

[0116] Hydrolyzing agents that can thus be used to cleave theoligonucleotide from the substrate are well known to those skilled inthe art and include hydroxide ion (e.g., as an aqueous solution ofsodium hydroxide), CH₃NH₂ or preferably, concentrated aqueous NH₄OH. Ina preferred embodiment, the entire substrate to which theoligonucleotide, having phosphate groups, is bonded is rinsed with asolution of the hydrolyzing agent. Alternatively, the hydrolyzing agentcan be dispensed as a microdroplet; in such a case, the microdropletpreferably comprises a solvent described in Section 5.2.

[0117] Where the nucleoside, as the first chemical species, is attachedto the substrate, or linker of the substrate, via an ester bond, it willbe understood that prior to reaction with an additional nucleoside, thefirst added nucleoside is deprotected with a deprotecting agent whichremoves a protecting group from its 5′ position. The subsequentlydispensed nucleoside, having a phosphoramidite group at its 3′ position,reacts with the nucleoside attached via the ester bond to the substrateor linker of the substrate, and having a deprotected hydroxyl group atits 5′ position, to form a phosphite group. The resulting phosphitegroup is than treated with an oxidizing agent, described above, to forma phosphate group. Successive iterations of deprotection, treatment witha phophoramidite functionalized nucleoside, and oxidation, elongate theresulting oligonucleotide chain.

[0118] In a different specific embodiment, a linker, attaching the firstchemical species' nucleoside unit to the substrate, contains a proteaserecognition site that, after synthesis of the oligonucleotide, iscleaved by use of the protease to release the substrate-anchoredoligonucleotide. The entire substrate is preferably rinsed with areaction mixture containing the protease; alternatively, the proteasecan be delivered as a microdroplet solution to the desired location onthe substrate where the oligonucleotide is tethered.

[0119] The resulting cleaved oligonucleotide is preferably soluble inthe solution of hydrolyzing agent or protease, as the case may be. Wherethe solution of hydrolyzing agent or protease, in which the substratecan be immersed, is contained in a vessel, the vessel will contain thecleaved oligonucleotide upon immersion of the oligonucleotide-anchoredsubstrate into the hydrolyzing agent or protease solution. Methods forisolating and purifying the cleaved oligonucleotide are well known tothose skilled in the art, and include, but are not limited to, gelelectrophoresis and high-performance liquid chromatography.

[0120] The isolated and/or purified oligonucleotide obtained by theabove methods can be used as known in the art, e.g., in hybridizationassays, for expression analysis or genotyping; as sequencing orpolymerase chain reaction (PCR) primers; or as templates for synthesisof oligonucleotide probes, etc.

[0121] It is to be understood that while the preferred method ofsynthesis of an oligonucleotide is in the 3′-5′ direction, the presentinvention also provides methods for synthesizing oligonucleotides in the5′-3′ direction. Oligonucleotides produced having this direction areuseful for enzymatic reactions, such as polymerization via DNApolymerase, while remaining attached to a substrate.

[0122] In the instance of synthesizing an oligonucleotide in the 5′-3′direction, a nucleoside having an hydroxyl protecting group at its 3′position, and a phosphoramidite at its 5′ position, is attached to thesubstrate. Preferably, and alternatively, a nucleoside having anhydroxyl protecting group at its 3′ position, and an activatedO-succinate group at its 5′ position is attached to the substrate. Thenucleoside having an hydroxyl protecting group at its 3′ position, and aphosphoramidite or an activated O-succinate group at its 5′ position, ispreferably applied to the substrate in a microdroplet of solution,preferably from an inkjet nozzle. Where the nucleoside has aphosphoramidite at its 5′ position, the substrate used has an hydroxylgroup, which reacts with the nucleoside's 5′ phosphoramidite group toform a phosphite group, which can then be converted to a phosphategroup. Where the nucleoside has an activated O-succinate group at its 5′position, the substrate used has an amino group, more preferably anamino group in the form of a long chain alkylamine, which reacts withthe nucleoside's 5′ activated O-succinate group to form an amide bond.Where the nucleoside has a phosphoramidite group at its 5′ position, theesterification reaction between the phosphoramidite group and thehydroxyl group of the substrate is facilitated by a catalyst, asdescribed above.

[0123] Once the nucleoside is anchored to the substrate, the 3′protecting group is removed as described above for the analogous 5′protecting group, preferably by rinsing the substrate with deprotectingagent, so as to expose a 3′ hydroxyl group. Then, a second nucleosidehaving a phosphoramidite, preferably having a cyanoethyl group, at its5′ position, and having a protecting group at its 3′ position, isdispensed, as a microdroplet of solution, at the locus of the substratewhere the first nucleoside was added. Following dispensation of thesecond nucleoside, a catalyst, such as one described above, isdispensed, preferably as a microdroplet of solution, at the locus of thesubstrate where the second nucleoside was added, to facilitate couplingbetween the first and second nucleosides. The reaction between the 3′hydroxyl group of the first nucleoside and the 5′ phosphoramidite groupof the second nucleoside forms a phosphite group, which is oxidized asdescribed above to form a phosphate group. Where the substrate has ahydroxyl group that reacts with a nucleoside's 5′ phosphoramidite group,it may be desirable to “cap” remaining substrate hydroxyl groups, asdescribed above, before proceeding to the subsequent steps.

[0124] Successive iterations of deprotection, dispensation of anadditional nucleoside, dispensation of catalyst and oxidation steps,elongates the oligonucleotide chain. Then, as described above, thecyanoethyl groups of the resulting oligonucleotide are removed. Finally,the resulting oligonucleotide is hydrolyzed from the substrate, using ahydrolyzing agent described above.

[0125] In addition, the present invention provides syntheses ofoligonucleotides having 5′-5′ or 3′-3′ linkages. Oligonucleotides havingthese linkages are useful for antisense and structural studies. Sucholigonucleotides are obtained according to the general methods above,and using a combination of nucleosides having an hydroxyl protectinggroup at the 5′ position and a phosphoramidite group at the 3′ position,and vice versa. For example, a nucleoside having a deprotected hydroxylgroup at its 5′ position that is anchored to a substrate via thenucleoside's 3′ group can react, preferably in the presence of acatalyst, with a second nucleoside having a phosphoramidite group at its5′ position and a protecting group at its 3′ position, to form a 5′-5′linkage. The resulting phosphite group is oxidized to a phosphate group,and the protecting group from the second nucleoside's 3′ position isremoved. Similarly, a third nucleoside having a phosphoramidite group atits 3′ position and a protecting group at its 5′ position can react,preferably in the presence of a catalyst, with the exposed 3′ hydroxylgroup of the second nucleoside to form a 3′-3′ linkage. Once theresulting phosphite group is oxidized to a phosphate group, thesynthesis can be continued using a nucleoside having either aphosphoramidite group at its 5′ position and a protecting group at its3′ position, or a phosphoramidite group at its 3′ position and aprotecting group at its 5′ position, depending upon the type of linkagedesired. Where it is desired that the resulting oligonucleotide becleaved from its substrate, the substrate preferably has an amino group,more preferably an amino group in the form of a long chain alkylamine,that reacts with a first nucleoside that has an activated O-succinategroup at its 3′ position and a protecting group at its 5′ position, oran activated O-succinate group at its 5′ position and a protecting groupat its 3′ position.

[0126] In yet another embodiment, the invention provides a method forobtaining oligonucleotides, having 3′-5′, 5′-3′, 3′-3′ or 5′-5′linkages, using the H-phosphonate method for oligonucleotide synthesis(see, for example, chapter 6 of J. F. Ramalho Ortigão et al.,Introduction to Solid-phase Oligonucleotide Chemistry(http://www.interactiva.de/oligoman/intro_inh.html)). In this instance,where the oligonucleotide to be synthesized is ultimately sought to becleaved from the substrate, a substrate having an amino group is reactedwith a nucleoside having a protecting group at its 5′ position and anactivated O-succinate group at its 3′ position, or having a protectinggroup at its 3′ position and an activated O-succinate group at its 5′position; where the oligonucleotide is not to be subsequently cleavedfrom the support, the support can have an hydroxyl group, which isreacted, preferably in the presence of a catalyst, with a nucleosidehaving a protecting group at its 5′ position and a phosphoramidite groupat its 3′ position, or having a protecting group at its 3′ position anda phosphoramidite group at its 5′ position. It is to be understood thatthe nucleoside reagents are delivered as microdroplets of solutions,preferably from inkjet nozzles.

[0127] Following removal of the protecting group, which exposes areactive hydroxyl group, a second nucleoside, having an H-phosphonatesalt group at its 5′ position and a protecting group at its 3′ position,or having an H-phosphonate salt group at its 3′ position and aprotecting group at its 5′ position, is dispensed as a microdropletsolution at the locus of the support at which the first nucleoside wasadded. Useful H-phosphonate salts are those that are soluble in thesolvents discussed in Section 5.2 above; preferably, the H-phosphonatesalts are triethylammonium salts, or salts of1,8-diazabicyclo[5.4.0.]undec-7-en (DBU). The reaction product of theH-phosphonate salts and the exposed hydroxyl group is an H-phosphonatediester.

[0128] Advantageously, the H-phosphonate salts react with the exposedhydroxyl group of the substrate-bound nucleoside in the presence of anactivator which, without being bound to any particular theory, isbelieved to increase the electrophilicity of the H-phosphonate group.Suitable activators include but are not limited to acid chlorides,preferably pivaloyl chloride and 1-adamantane carbonyl chloride; andanhydrides, preferably dipentafluorophenyl carbonate. The activators canbe dispensed as a microdroplet with the H-phosphonate salts as part ofthe same solution, or can be dispensed as separate microdroplets fromseparate solutions. Successive dispensations of H-phosphonatesalt/activator solutions as microdroplets, or successive iterations ofseparate H-phosphonate salt and activator dispensation steps, elongatesthe resulting oligonucleotide chain.

[0129] Once the oligonucleotide has reached its desired length, theH-phosphonate diester linkages are oxidized using conventional reagents,preferably an aqueous iodine solution, to afford phosphate groups. Theoxidizing agent can be dispensed as a microdroplet comprising a solventdescribed in Section 5.2; alternatively, the entire substrate to whichthe oligonucleotide is attached can be washed with the oxidizingreagent. The oligonucleotide can then be cleaved from the substrateaccording to methods described above.

[0130] In addition to the chemistries described above, alternativereactions can be used in the methods of the invention where oligomerscomprising modified nucleosides or nucleoside derivatives aresynthesized. Such modified nucleosides include, for example,combinations of modified phosphodiester linkages such asphosphorothioate, phosphorodithioate and methylphosphonate, as well asnucleosides having such modified bases such as inosine, 5′-nitroindoleand 3′ -nitropyrrole.

[0131] Synthesis of oligoribonucleotides, e.g., RNA, can similarly beaccomplished using the present methods. Effective chemical methods foroligoribonucleotide synthesis have added complications resulting fromthe presence of the ribose 2′-hydroxyl group. However, ribonucleosidecoupling chemistries and protecting groups are available and well knownto those skilled in the art. Therefore, such chemistries are applicableto the methods described herein.

[0132] As with oligodeoxyribonucleotides described above, a range ofmodifications can similarly be introduced into the base, the sugar, orthe phosphate portions of oligoribonucleotides, e.g., by preparation ofappropriately protected phosphoramidite or H-phosphonate ribonucleosidemonomers, and/or coupling such modified forms into oligoribonucleotidesby solid-phase synthesis. Modified ribonucleoside analogues include, forexample, 2′-O-methyl, 2′-O-allyl, 2′-fluoro, 2′-amino phosphorothioate,2′-O-Me methylphosphonate, α-ribose and 2′-5′-linked ribonucleosideanalogs.

[0133] In another preferred embodiment, the first chemical species is anamino acid; the second chemical species is a substrate having reactivefunctional groups, a linker attached to a substrate, or an amino acid orpeptide attached to either the linker or directly to the substrate; andthe third chemical species is an amino acid or a peptide chemicallyattached to either the linker or directly to the substrate. In thisembodiment, the first chemical species is an amino acid having aprotecting group on the carboxy group of its carboxy terminus, and thesecond chemical species is (a) a substrate, or a linker attached to asubstrate, having an electrophilic group that is capable of forming astable covalent bond with the amino group of the amino terminus of theamino acid, or (b) an amino acid or peptide attached to either thelinker or directly to the substrate, and having a carboxy terminus thatis capable of forming an amide bond with the amino group of the aminoterminus of the amino acid. Alternatively and preferably, the firstchemical species is an amino acid having a protecting group on the aminogroup of its amino terminus, and the second chemical species is (a) asubstrate or a linker attached to a substrate having a nucleophilicgroup that is capable of forming a stable covalent bond with the carboxyterminus of the amino acid, or (b) an amino acid or peptide attached toeither the linker or directly to the substrate, and having an aminoterminus that is capable of forming an amide bond with the carboxyterminus of the amino acid. It will be understood that if the firstchemical species and second chemical species bear additional reactivegroups, those reactive groups can have additional protecting groups, soas to preclude unwanted side reactions with those groups if notprotected.

[0134] Advantageously, where peptides are sought to be obtained, thereaction between the first chemical species and the second chemicalspecies takes place in the presence of a catalyst; preferably, astoichiometric amount of a catalyst such as dicyclohexylcarbodiimide orthe like. In such a case the microdroplet solution can comprise thecatalyst as well as the first chemical species.

[0135] Typically, a solution comprising an amino acid having aprotecting group on the amino group of its amino terminus as the firstchemical species and a stoichiometric amount of dicyclohexylcarbodiimidecatalyst, is dispensed as a microdroplet onto a second chemical species,e.g., a substrate with a linker having, for example, hydroxyl functionalgroups, thereby forming a microdot containing an amino acid covalentlybonded to the linker of the substrate via an ester bond as the thirdchemical species.

[0136] It is to be pointed out that suitable protecting groups for theamino group of the amino terminus of an amino acid includetert-butoxycarbonyl (tBOC) and 9-fluorenylmethoxycarbonyl (FMOC)protecting groups, and other protecting groups disclosed in Theodora W.Greene, Protecting Groups in Organic Synthesis 218-49 (1981),incorporated herein by reference.

[0137] The resulting N-protected amino acid that is covalently bonded tothe linker of the substrate is then treated with a deprotecting agentthat can remove the protecting group from the amino group of the aminoacid's amino terminus: in the case where a tBOC protecting group isused, the deprotecting agent is an acid such as HCl or trifluoroaceticacid; in the case where an FMOC protecting group is used, thedeprotecting agent is an organic base such as piperidine, morpholine orethanolamine. The protecting group can be removed by immersing orotherwise washing the substrate in a bath or stream of a solution of thedeprotecting agent. Alternatively, the deprotecting agent can be added,in the form of a microdroplet, onto the N-protected amino acid that iscovalently bonded to the linker of the substrate. In such a case, themicrodroplet preferably comprises a solvent described in Section 5.2.

[0138] The resulting deprotected amino acid that is covalently bonded tothe linker of the substrate, becomes the second chemical speciesrelative to an impinging microdroplet of a solution of either the sameor a different amino acid having a protecting group on the amino groupof its amino terminus, and so on. The entire process is repeated until apeptide having a desired sequence or length is obtained.

[0139] It is to be pointed out that this process may be repeated atdifferent loci of the substrate, using different first chemical speciesand second chemical species, so as to obtain, if desired, a differentpeptide at each loci.

[0140] The resulting peptide which is covalently attached to the linkerof the substrate, and which has a protecting group on the amino group ofits amino terminus, is treated, either via submersion of the substrateor via microdroplet impingement as described above, with a deprotectingagent that removes that protecting group and preferably all of theremaining protecting groups on the peptide, if any. If protecting groupsremain on the peptide subsequent to treatment with the deprotectingagent, subsequent treatments with deprotecting agents can be effecteduntil all of the protecting groups have been removed.

[0141] If desired, the resulting deprotected peptide synthesized by theabove method can then be cleaved from the linker using conditions thatwill hydrolyze an ester bond in the presence of an amide bond, includingtreatment with mild hydroxide base, as well as other suitable conditionsknown to those skilled in the art.

[0142] The methods of the invention can be applied to other chemistriesthat rely on iterations of coupling and deprotection. For example, usingthe present methods, it is possible to construct arrays of otherheteromeric polymers with sequence dependent properties.

[0143] It will be realized that a particular advantage of the method ofthe invention is that by keeping a record of the first chemical speciesdispensed, and accordingly third chemical species formed, at each of themicrodot loci, libraries of chemical compounds having known sequencescan be easily obtained. Such chemical compounds can have a variety ofuses including, but not limited to, screening for biological activitywhereby the respective chemical compound at each locus is exposed to alabeled or unlabeled nucleic acid or receptor, such as an antibody, acell receptor, or any other variety of receptor.

[0144] The following examples are presented by way of illustration andnot by limitation on the scope of the invention.

AUTOMATED SYNTHESIS SYSTEM

[0145] The methods of the invention for chemical synthesis usingmicrodroplets are preferably automated. Preferably, the methods areautomated as described below, using the exemplary apparatus and softwareas described herein.

SYSTEM IMPLEMENTATION

[0146] Shown in FIG. 5 is a preferred embodiment of an automated systemfor large-scale synthesis of biopolymers in accordance with theinvention. As used herein, the term biopolymer is intended to mean anyof numerous biologically occurring compounds which are synthesized fromtwo or more individual monomer building blocks. Nucleic acids,polypeptides and carbohydrates are specific examples of biopolymers. Theindividual monomers for these biopolymers consist of nucleosides, aminoacids and sugars, respectively. The term is intended to include naturaland non-naturally occurring monomers as well as derivatives, analogues,and mimetics thereof.

[0147] The automated system is designated by reference numeral 20.Generally, system 20 comprises scanning transport 22, treating transport23, a print head assembly 24, an alignment unit 26, a transfer station28, a flow cell 30, and a substrate storage rack 32. The components aremounted, for example, on a base 34, and enclosed by a cover (not shown)so that processing can be performed in a dry nitrogen environment.

[0148] The components are used to manipulate a planar substrate and tosynthesize biopolymers on the substrate under the automated control of acomputer. The substrate used for the synthesis of two-dimensionalbiopolymer arrays is generally a wafer having a flat planar surfacewhich has, or can be modified to have, reactive groups suitable forattaching further organic molecules. The substrate can additionally beporous so long as it supports the synthesis of biopolymer arrays.Specific examples of substrates useful in the automated system of theinvention include glass, silica, silicon, polypropylene, TEFLON®,polyethylimine, nylon, fiberglass, paper and polystyrene. The surfacecan additionally consist of bead structures attached to a solid surface,wherein the beads are composed of one or more of the precedingmaterials. The dimensions of the substrate can vary and are determinedto be complementary to the supporting structures of the automatedsystem. The dimensions can be altered depending on the desired size andapplication of the array and the design of the supporting structureswhich hold the substrate.

[0149] The substrate is cycled once over the print head assembly to makea single deposit of a chosen biopolymer monomer at each desired site. Inthis single cycle, different sites can receive different monomers. Forthe synthesis of nucleic acid biopolymers, for example, any one of thefour monomers is available for any particular site during any singleprint head cycle. A catalyst is applied by the print head to eachsubstrate site after the monomers are deposited.

[0150] After a print head cycle, treating transport 23 is used to movethe substrate from the print head assembly to flow cell 30, which“treats” the substrate by exposing it to selective fluids in order torinse off unconnected monomers, oxidize, and deprotect the substrate.Once rinsed, the substrate is moved again to print head assembly 24 fora further cycle of monomer deposits, and then rinsed again in the flowcell. These steps are repeated numerous times to build desiredbiopolymer sequences. Different biopolymer sequences can be assembled ateach site by using different sequences of monomers.

[0151] Inkjet printers generally employ print heads that may contain 50to 100 independently controlled nozzles. With each nozzle operating atseveral hundred cycles per second (Hertz or Hz), a machine with fivesuch print heads can deliver the appropriate reagents to 100,000 wellsin a matter of seconds. A complete synthesis cycle can take, forexample, 5 minutes, or just over 2 hours for an array of 100,000biopolymers having 25 monomer residues. Print heads having more or lessnozzles and which operate at different speeds can be used as well.Additionally, multiple print heads can be simultaneously used tosynthesize the biopolymer arrays. Such configurations are known to thoseskilled in the art and will vary depending on the size, format andintended use of the array and the different reagents and monomers to bedeposited.

[0152]FIG. 6 shows print head assembly 24. The print head assemblycomprises two print heads 36, mounted within an aluminum block 38. Thepreferred print heads are inkjet print heads by Epson America, Inc., ofTorrance, Calif., sold as spare parts for use in STYLUS COLOR II™ inkjet printers. These print heads are intended for use in depositing apattern of ink droplets onto media positioned adjacent the print heads.More specifically, each print head comprises an array of 60 individualnozzles, which are piezoelectric pumps created with known etchingtechniques and formed with small cavities with narrow inlets andnozzles, as explained with respect to FIG. 3.

[0153] In this embodiment, the two print heads are aligned with eachother and directed upwardly, to deposit liquid on a substrate that ispositioned over the print heads. Block 38 and print heads 36 aresupported on base 34 (FIG. 5) by calibration devices 39, which includeadjustments for height, rotation, pitch, and yaw. Calibration devices 39allow the print heads to be precisely aligned with the mechanism,described below, that positions substrates over the print head.

[0154] Each of the print heads has three separate fluid manifolds,attached to the manifold inlets. When combined, the two print heads havesix manifolds, allowing the use of six different reagents. Externalreservoirs 40 (FIG. 5) are connected to supply reagents to themanifolds. Each print head has 60 nozzles organized as 3 banks of 20nozzles. The 20 nozzles in a bank have a common reagent manifold. Eachbank of nozzles is arranged linearly, along an axis that isperpendicular to the direction in which the substrate is to be movedacross the print heads.

[0155] In the specific embodiment directed to the synthesis of nucleicacid biopolymers, four manifolds contain different nucleoside monomersas reagents. The monomers can be mixed with a catalyst such as5-methylthiotetrazole, tetrazole, or preferably 5-ethylthiotetrazole, inadvance or, alternatively, another manifold can be used to contain andapply the catalyst.

[0156] A complete synthesis cycle starts by delivering the appropriatenucleoside monomers along with a catalyst such as 5-ethylthiotetrazoleto the substrate. After a layer of monomers are deposited on thesubstrate, the entire substrate is treated by rinsing off excessmonomers, exposing the substrate to an oxidizing solution and thendeprotecting for the next round of synthesis. The rinses are common toall the loci on the substrate and can be done, for example, by bulkimmersion. One such cycle adds one monomer to each oligonucleotide, thusa substrate of oligonucleotides having a length of ten nucleosidesrequires 10 such cycles.

[0157] The number of cycles, and therefore, the length of the biopolymerwill be determined by the need and desired use of the array. As such,the biopolymer lengths which can be achieved using the automated systemof the invention are only limited by the types of reactive chemicalspecies and existing coupling chemistries. For nucleic acid biopolymers,oligonucleotides of unlimited length, preferably between 10 and 100monomers in length, and more preferably between 20 and 60 monomers inlength, can routinely be synthesized.

[0158] For use in the automated synthesis system of the invention, thebiopolymer monomers may be either dissolved in a solvent or,alternatively, the automated system can be adapted to contain a mixingreservoir to supply a solvent. Thus, in the automated system shown inFIG. 5, one or more of the external reservoirs 40 can contain a solventor monomers dissolved in a solvent. The solvent is preferably one of thesolvents described in Section 5.2.

[0159] Scanning transport 22 is used to “scan” a substrate by moving thesubstrate over print head assembly 24 for depositing nucleoside monomersat specified loci or sites on the substrate. While print head control isaccomplished in a manner similar to that commonly employed with inkjetprinters, unlike in a standard inkjet printer the substrate is movedrather than the print head assembly itself. As shown in FIG. 7, thescanning transport comprises a translational stage having at least twoaxes of linear movement. More specifically, the scanning transport is anX-Y translation stage 41 oriented to provide two degrees of horizontalmotion. Movement along each axis is accomplished by an electronicstepping motor that is geared to provide a linear resolution of about 5μm. The preferred system uses an X-Y translation stage from ParkerHannifin Corp, Model 310062AT.

[0160] To hold the substrate, scanning transport 22 includes a vacuumchuck 42. Vacuum chuck 42 is mounted at the end of scanning arm 44 thatextends laterally from X-Y translation stage 41. The vacuum chuck isconnected relative to the X-Y translation stage so that the vacuum chuckcan be moved back and forth and sideways over the print head.

[0161] The vacuum chuck includes a circular plate 46 having a planarlower surface with a plurality of interconnected concentric grooves (notshown). Vacuum is selectively applied to the interconnected grooves tohold the substrate to the lower surface of the vacuum chuck. To applyvacuum, a vacuum tube (not shown) extends to the grooves from anexternal vacuum source that is controlled by a solenoid valve (notshown). Circular plate 46 is mounted for rotation within a mated openingin substrate holder 47 that is in turn attached to a distal end ofscanning arm 44. The opening preferably has a lower lip to support thecircular plate 46 by its periphery from beneath. Clips (not shown) canbe used to retain the circular plate in its mated opening, and toprovide moderate friction that prevents accidental rotation of plate 46.

[0162] A small rotational adjustment pin 48 extends radially outwardfrom the circular plate, beyond substrate holder 47. The rotationaladjustment pin can be engaged to rotate circular plate 46 about avertical axis.

[0163] The rotation feature of circular plate 46 is used to rotationallycalibrate a substrate relative to the print head. No calibration isnecessary for a substrate that is about to undergo its first print headcycle because initial positioning of the substrate with respect to thescanning transport is used to establish an initial pattern of synthesissites on the substrate. During subsequent cycles, however, the substratemight be positioned differently on the vacuum chuck, requiringcalibration steps. Horizontal position differences in position can becompensated for by translational stage 41 and its controllingelectronics. Rotational misalignment (about the vertical z axis) iscorrected by rotating circular plate 46 within its substrate holder 47.Specifically, the scanning transport 22 is moved to engage rotationaladjustment pin 48 against a stationary vertical reference pin (notshown) mounted next to the alignment unit 26. In this fashion, rotatingcircular plate 46 can be rotated by an amount that restores its originalrotational alignment.

[0164] The amount of existing translational and rotational misalignmentis determined by alignment unit 26. FIG. 8 shows this unit in moredetail. Alignment unit 26 comprises a marker 50 and a camera 52. Marker50 can be activated to establish marks at particular loci on thesubstrate for positionally calibrating the substrate relative to thescanning transport and to the print head assembly. It comprises adiamond tip or point that can be raised and lowered in response toactivation and deactivation of a solenoid 54. When the marker is raised,it contacts an adjacent substrate. If the substrate is moved withrespect to the marker, the marker scratches or scores the substrate,resulting in a visible line.

[0165] The marker is mounted at an intermediate position along apivoting element 56 that is mounted at one end 57 for pivoting about ahorizontal axis. Solenoid 54 has a vertically movable plunger 58 thatengages the pivoting element at its other end 59.

[0166] Camera 52 comprises a lens unit 60 and a charged coupled device(CCD) imaging element 61 that are used to positionally calibrate thesubstrate relative to the scanning transport and to the print headassembly. Marker 50, pivoting element 57, solenoid 54, and camera 52 aremounted to a block 62 that can be adjusted vertically by means of amicrometer adjustment 63. This adjustment is used to focus the lens andCCD combination on an adjacent substrate. The preferred system uses acamera from Polaris Industries, Model MB-810B Micro Size CCD.

[0167] In use, the initial positioning and alignment of a substrate isrecorded by scoring two marks on the substrate. Preferably, a cross or Xis made on two opposite ends or corners of the substrate. Duringsubsequent handling of a particular substrate, each mark is positionedover lens 60 and its precise position is recorded. This information isused to calculate horizontal correction factors in the X and Ydirections, and to calculate rotational misalignment. The horizontalcorrection factors are used when positioning the substrate over theprint head with the scanning transport 22. The rotational misalignmentis corrected by rotating circular plate 46 within its substrate holder47 as described above.

[0168]FIG. 9 shows flow cell 30. Flow cell 30 is adapted for receivingthe substrate and for “treating” the substrate by exposing the substrateto one or more selected reagents. Specifically, it is used for washingoff unattached monomers, exposing the substrate to an oxidizingsolution, and deprotecting the terminal nucleoside of theoligonucleotides being formed for the next round of synthesis.

[0169] In a preferred embodiment, flow cell 30 includes a rectangularlyshaped stationary plate 70 mounted perpendicularly to base 34. A squarebacking plate 76 which is oriented parallel to stationary plate 70 isfixed to stationary plate 70 with four cylindrical rods 77. A squaremoving plate 72 that is parallel to and located between stationary plate70 and backing plate 76 moves back and forth between these fixed platesguided by the rods 77. Each rod 77 fits through a hole located near acorner of moving plate 72. The holes are sized to rods 77 for a closesliding fit. One end of each rod 77 is fixed near a corner of backingplate 76. The other end of each rod 77 is fixed to stationary plate 70.When moving plate 72 moves toward stationary plate 70, a substrate issandwiched between the two plates. Moving plate 72 is driven by apneumatic cylinder 74 whose longitudinal axis is parallel to thedirection of travel of moving plate 72. The base of pneumatic cylinder74 is fixed to backing plate 76 and the end of piston rod 79 ofpneumatic cylinder 74 is fixed to moving plate 72. Moving plate 72 isguided by the rods 77 to slide toward and away from stationary plate 70in response to activation of pneumatic cylinder 74.

[0170] A vertical surface 80 of stationary plate 70 which faces movingplate 72 has a raised circular ring 82 made of a material that canwithstand contact with the solvents used to treat the substrate. Theraised circular ring 82 is sufficiently large in diameter to surroundall portions of a substrate upon which reagents have been deposited. Aninlet 83 extends through stationary plate 70 just inside the raisedcircular ring 82 at its lowermost portion and an outlet 84 extendsthrough the stationary plate 70 just inside the raised circular ring 82at its uppermost portion.

[0171] The planar surface of moving plate 72 facing stationary plate 70has embedded in it a rubber o-ring (not shown) which protrudes above thesurface of moving plate 72 and can press a substrate against raisedcircular ring 82. The rubber o-ring is the same diameter as the circularring 82 so as to directly transfer pressure to the surface of thecircular ring 82 and not to crack the substrate that is held between theo-ring and circular ring 82. A substrate so pressed against raisedcircular ring 82 forms a sealed chamber that is bounded by the surfaceof the substrate, by vertical surface 80, and by raised circular ring82. The surface of the substrate forming a portion of the chamber can beexposed to various solvents by injecting such solvents into the chamberthrough inlet 83. The solvents exit the chamber through outlet 84. Thisaspect of the invention is automated by utilizing solenoid controlledvalves in conjunction with solvent containers and appropriate tubing(not shown).

[0172] Treating transport 23 which is used for placing a substratewithin the flow cell 30 comprises an X-Y translation stage, an elevator86 that provides vertical (Z axis) movement, and a rotator 87 to providemotorized rotational movement about the longitudinal axis of anelongated rod 90 which extends from rotator 87. Movement along each ofthe X, Y, Z, and rotational axes is controlled by a stepping motor. Thetreating transport 23 also includes a vacuum chuck 91 which is attachedto the end of the elongated rod 90 distal from the rotator 87. Thevacuum chuck 91 has a circular shape that is approximately the size ofthe substrate upon which synthesis is being performed. The vacuum chuck91 is thus configured to hold the surface of the substrate away from thesurface on which reagents are being deposited. The vacuum chuck 91 isrelatively thin so that it can be positioned conveniently betweenstationary plate 70 and moving plate 72 of flow cell 30. By controllingthe X-Y translation stage, the elevator 86, and the rotator 87, vacuumchuck 91 can be moved along two horizontal axes and a vertical axis, andcan also be rotated about one of the horizontal axes.

[0173] When vacuum chuck 91 positions a substrate between circular ring82 and the surface of moving plate 72, a low vacuum of approximatelythree feet of water (1.3 pounds per square inch) is created within thechamber formed by the substrate, vertical surface 80, and circular ring82. This slow vacuum holds the substrate in place after the vacuum chuck91 retracts and before moving plate 72 moves in to firmly press thesubstrate against circular ring 82.

[0174] Transfer station 28, shown in more detail in FIG. 10, serves anintermediate holding location for the substrate when the substrate istransferred between scanning transport 22 and treating transport 23.Transfer station 28 includes a planar motorized platform 93 orientedparallel to base 34 that supports a planar vacuum chuck 94. Vacuum chuck94 has a square upper surface oriented parallel to motorized platform 93upon which a substrate can rest. Vacuum is applied about the peripheryof the upper surface of vacuum chuck 94 to secure the substrate when thesubstrate is placed on vacuum chuck 94.

[0175] Vacuum chuck 94 is supported on top of motorized platform 93 byfour coil springs 95 which are located between the motorized platform 93and the vacuum chuck 94. One coil spring 95 is positioned near each ofthe corners of vacuum chuck 94. Motorized platform 93 can be raised andlowered by a stepping motor 96 which is located below motorized platform93. Vacuum is communicated to vacuum chuck 94 by a vacuum line 97, whichcommunicates the vacuum by a solenoid controlled valve (not shown). Toreceive a substrate held by vacuum chuck 42 of scanning transport 22,the motorized platform 93 is raised until the upper surface of vacuumchuck 94 contacts the lower surface of the substrate. Motorized platform93 does not have to move vertically to transfer a substrate to or fromvacuum chuck 91 of treating transport 23 since that mechanism hasvertical movement capability. Coil spring 95 absorbs any over-travel ofmotorized platform 93. Once the substrate has been grasped by vacuumchuck 94, motorized platform 93 is lowered.

[0176]FIG. 11 shows control components used to manipulate the variouselectromechanical components described above. Such components include acomputer 100 having a microprocessor and associated memory componentssuch as electronic memory and mass storage devices. The preferred systemuses an IBM-compatible computer. Computer 100 includes common userinterface components such as a monitor, a keyboard, and a mouse. Thecomputer also has an expansion bus allowing various specializedperipheral devices and interfaces to be used in conjunction with thecomputer.

[0177] Various electronic hardware is provided for use in conjunctionwith computer 100 for actuating solenoids, stepping motors, and othercomponents that control the physical operation of the hardware describedabove. Some of these components are implemented on expansion cards thatare plugged directly into the expansion bus of computer 100, while othercomponents are external to computer 100. The specific design andconfiguration of these electronic components will vary depending uponthe particular electromechanical components used. As an example, thecontrol components of FIG. 11 include a digital I/O card 102 having aplurality of digital inputs and outputs. This card is plugged directlyinto the expansion bus of computer 100. External driver circuits 104 areused as a buffer between the computer-level signals of I/O card 102 andthe higher level signals used by the electromechanical componentsthemselves. Solenoids are controlled with outputs from I/O card 102.

[0178] A frame capture circuit 110 is plugged into the expansion bus ofcomputer 100. Frame capture circuit 110 receives a video signal fromcamera 52 and provides a two-dimensional array of pixel values for useby computer 100. Frame capture circuit 110 and the digital image itproduces are used to locate the substrate marks made by marker 50 and tothereby determine any necessary compensation in positioning thesubstrate with respect to the print head. The preferred system uses aframe capture circuit on a WinVision Video capture board from QuantaCorp.

[0179] A plurality of motion control cards 106 are also plugged into theexpansion bus of computer 100. These are conventional stepping motorcontrol cards that operate in conjunction with computer 100 to controlmovements of the various stepping motors described above. The preferredsystem uses motion control cards from Oregon Micro Systems Inc., ModelPC34-4. External driver circuits or amplifiers 108 are electricallyconnected between the motion control cards and the stepping motorsthemselves.

[0180] A print head controller 109 is also plugged into the expansionbus of computer 100. This circuit has electrical drivers that areconfigured specifically for the particular print heads that are chosenfor use in print head assembly 24. In many cases, it will be necessaryfor these drivers to receive position feedback signals from the motioncontrol circuits controlling the scanning transport 22, in order tocoordinate print head firing with progress of the substrate across theprint head assembly.

[0181]FIG. 12 shows in detail printer head controller 109 forcontrolling the inkjet printer heads. Trigger RAM 201 stores X-positionsof the substrate where the deposition is to take place. Quadraturedecoder & counter 202 produces the current X-position of the substrateby decoding the signal from a stepping motor used to move the substrate.Equality test 203 compares the current X-position with the X-position ofdeposition and produces a match signal. The match signal is provided totiming logic 204, which generates various timing signals to synchronizethe activities across the components. Timing logic 204 uses 16-bitcounter 205 to generate address signals to access the trigger RAM 201 aswell as spit vector RAM 206. The spit vector RAM 206 stores a bit mapfor each trigger point where each bit represent the activation of anozzle. The bitmap is loaded to each head using parallel-load shiftregisters 207 and 208. Timing logic 204 also uses 16-bit counter 209 togenerate an address signal to access waveform RAM 210, which containsdata representing the electric pulse waveform supplied to the printhead. The waveform data are loaded to 8-bit latches 211 and 212 andconverted to pulse signals using digital-to-analog (D-to-A) converters213 and 214.

[0182] Computer 100 is programmed using conventional programmingtechniques to control movement of the various moving parts describedabove. Other types of computers or control logic could of course be usedin place of the computer described. For example, an industrial-controlcomputer unit referred to as a programmable controller might besubstituted in place of a desktop computer.

[0183] Computer 100 is programmed specifically to move substratesbetween rack 32 and the two processing components: print head assembly24 and flow cell 30. With respect to a single substrate, a first stepmight comprise retrieving the substrate from rack 32 with treatingtransport 23 and moving the substrate to transfer station 28. Rack 32has slots for receiving and storing substrates in vertical orientations.Other orientations can be equally substituted. Since substrates arestored vertically in rack 32, vacuum chuck 90 of treating transport 23is turned to a vertical orientation and moved adjacent the rear surfaceof the substrate. Vacuum is applied to vacuum chuck 91 by activating asolenoid valve, and vacuum chuck 91 is withdrawn from rack 32 along withthe substrate.

[0184] Vacuum chuck 91 is then rotated to a horizontal orientation andmoved to a position over transfer station 28. Vacuum chuck 91 is loweredto place the substrate on vacuum chuck 94. Vacuum is applied to vacuumchuck 94 of transport station 28 by activating a solenoid valve. Thevacuum is disconnected from vacuum chuck 91.

[0185] Vacuum chuck 42 of scanning transport 22 is then moved over thesubstrate, and the substrate is raised by transfer station 28 so that itengages vacuum chuck 42. Vacuum is applied to vacuum chuck 42 byactivating a solenoid valve.

[0186] If this is the initial cycling of the substrate, it is moved overmarker 50 to establish one or more calibration marks on the substrate asalready described. The substrate is then moved over print head assembly24.

[0187] If the substrate has already been cycled over the print head, thesubstrate is moved over camera 52 while computer 100 performs a step oflocating the marks in conjunction with the camera. Accordingly, eachmark is located individually. That is, one of the two marks ispositioned within the camera's field of view and an image is acquired bycomputer 100. This is repeated with the other mark. Using the twoacquired images that include the marks, the computer determines theposition of the substrate relative to X-Y translation stage 41 and inrelation to its initial position as represented by the marks. Thecomputer then performs a software calibration of X-Y translation stage41 to account for any difference in the position of the substrate incomparison to its original position.

[0188] The computer also determines the rotational misalignment of thesubstrate with reference to the marks, again using the acquired images.In response to any rotational misalignment, the computer moves X-Ytranslational stage 41 to engage rotational adjustment pin 48 of thevacuum chuck 42 with the vertical reference pin to rotate the circularplate 46 of vacuum chuck 42 by an angular displacement that corrects forthe misalignment.

[0189] Once the substrate has been positionally calibrated, the computermoves the substrate over print head assembly 24 with scanning transport22 while simultaneously firing print head 36 repeatedly to deposit thenucleoside monomers at appropriate sites. Multiple passes might berequired to reach all the sites of the substrate. Further passes aremade to apply a catalyst. The computer then moves the substrate totransfer station 28 with scanning transport 22. Treating transport 23 isthen moved to transfer station 28 to pick up the substrate. Vacuum chuck91 of treating transport 23 carries the substrate to flow cell 30 andpositions it therein. A low vacuum of approximately three feet of water(1.3 pounds per square inch) is applied to the chamber formed by thesubstrate, vertical surface 80, and circular ring 82. This low pressureholds the substrate in place while the vacuum chuck 91 retracts. Movingplate 72 then clamps the substrate firmly in the flow cell 30. Rinsingsolvents are then cycled through flow cell 30. The substrate is thenreleased from the flow cell. If processing is complete, treatingtransport 23 moves the substrate back to rack 32. Otherwise, the stepsabove are repeated.

SOFTWARE IMPLEMENTATION

[0190] Flow charts detailing the operation of the software controllingthe automated system are depicted in FIGS. 13-21. Here, while “wafer” isused to describe a specific example of a substrate, it will be clearthat other substrates may also be used.

[0191]FIG. 13 shows in detail the steps for the initialization of theprogram. After the program starts at step 1000, it reads in the filestoring the pump driving waveforms describing voltage waveforms foractivating the piezoelectric pumps in the inkjet print head (step 1001).Next, the program reads in the file describing the print head geometrydescribing how the nozzles in the print head are spaced and the contentsof each manifold connected to the nozzles (step 1002). Next, the programinitializes the mapping from individual nozzles on the inkjet printheads to bits in the spit vector RAM (step 1004). The program then readsin a list containing the name of an oligo specification file storing thegeometry of the desired pattern to be deposited in a particular wafer tobe processed in a particular run (step 1005).

[0192] If the program is done with wafer-specific initialization, itproceeds to the main loop in step 1007. Otherwise, the program reads theoligo specification file storing the geometry of the desired pattern tobe deposited in a particular wafer (step 1008). The program thencalculates all trigger RAM 201 entries that will be used, which includea distinct inkjet nozzle trigger point (X-location) and a distinctcolumn of dots in the pattern on a wafer at each trigger point (step1009). The program then calculates all Y-positions (passes) that thescanning arm will need to make in the course of synthesizing one layerof nucleoside monomers (step 1010). During the operation, the scanningarm moves to Y-positions, then sweeps across the X-positions required totrip all the desired trigger points. The required Y-positions aredetermined by the number and spacing of the rows of dots in the desiredpattern and the space spanned by a column of inkjet nozzles on an inkjetprint head. The program also determines the number of times the triggerRAM 201 will need to be reloaded while scanning one layer of nucleosidemonomers. The program maps each row in the directed wafer pattern towhat will be the nearest row of nozzles during the appropriate Y-pass(step 1011).

[0193]FIG. 14 shows the main loop involving the operation of theautomated synthesis system. In the case where there are multiple flowcells, the program first determines whether all flow cells were checked(step 1100). If they were not, it checks each flow cell to see whetherit is done with treating the wafer (steps 1104-1106). If the treatmentis done, the wafer is transferred to scanning 44 arm if the scanning armis empty.

[0194] If all the flow cells were checked, the program checks whetherthere is a wafer on the scanning arm (step 1101). If there is, itproceeds to the Check_Alignment routine (step 1109) where it doesinitial positioning and alignment of the wafer. If there is no longer awafer on the scanning arm (i.e., it was removed during theCheck_Alignment routine) or there wasn't to start with (step 1111), theprogram checks whether the number of wafers in the system is less thanthe number of flow cells minus 1, i.e., whether all the flow cells arenot full. If all the flow cells are not full, the system loads the nextwafer from wafer rack 32 (steps 1102-1103).

[0195]FIG. 15 shows the Check_Alignment routine in detail. The programchecks whether the wafer had been aligned previously (step 1200) afterits most recent transfer to the scanning arm. If so, the program checkswhether the wafer is to receive the first layer of deposition (step1201). If it is, the program executes a routine for “tagging” the wafer,i.e., making registration marks for subsequent re-alignment (step 1202),which will be described in more detail with reference to FIG. 12. Theprogram then executes a routine for aligning the wafer (step 1203),which will also be described in more detail with reference to FIG. 12.

[0196] If the wafer had been aligned before, the program checks whetherthe wafer has been just aligned (step 1204). If so, the program checkswhether all the layers on the wafer are done (step 1205). If so, thewafer is transferred back to the wafer storage rack (step 1206).Otherwise, the program executes the Do_a_layer routine for depositing alayer on the wafer (step 1207). If the wafer has not been just aligned,i.e., the deposition has just been finished, the wafer is transferred toan empty flow cell (step 1209) and the treatment of the wafer starts(step 1209).

[0197]FIG. 16 shows in detail the routine for tagging the wafer and theroutine for aligning the wafer.

[0198] The routine for tagging the wafer by scoring registration marksconsists of steps 1300-1304. The rotational position of vacuum chuck 42is initialized by bumping rotational adjustment pin 48 against thevertical reference pin to return the adjustment pin to a known location(step 1300). The scanning arm moves the wafer to a location for thefirst registration mark on the wafer (step 1301). A cross is cut on thewafer by coordinating the movement of the scanning arm with theactivation of solenoid 54 for raising the scribe tip (step 1302).Scanning arm 44 moves the wafer to another location for the secondregistration mark (step 1303). Another cross is cut on the wafer (step1304).

[0199] The routine for aligning the wafer once the registration marksare scored on the wafer consists of steps 1305-1404. Vacuum chuck 42 isinitialized by bumping rotational adjustment pin 48 against the verticalreference pin to return the adjustment pin to a known location (step1305). Scanning arm 44 moves the wafer such that the first registrationmark will be centered over the center of camera 52 if the alignment wasalready correct (step 1306). This should place the registration marksomewhere in the camera's field of view. The center position of thecross of the first registration mark is measured (step 1307). Scanningarm 44 moves the wafer such that the second registration mark on thewafer will be centered over the center of the camera if the alignmentwas already correct (step 1308). The center position of the cross of thesecond registration mark is measured (step 1309). The program calculatesthe angle that the wafer is rotated away from a perfectly alignedposition from the measured positions of the two registration marks (step1400). The program then calculates the direction and the magnitude ofthe deflection of rotational adjustment pin 48 required to correct theabove rotation (step 1401). The rotational adjustment pin is bumpedagainst the vertical reference pin to correct the rotation (step 1402).The scanning arm moves the wafer so that the second registration mark isnow over the center of the camera (step 1403). The program executes theGo_home routine for calculating the X and Y-position adjustments suchthat the center of the registration mark is located directly over thecenter of the camera (step 1404).

[0200]FIG. 17 shows the Go_home routine in detail. The program firstchecks whether the second registration mark is at the home position,i.e., being centered over the center of the camera (step 1501). If it isnot, the position of the second registration mark is recorded and thescanning arm moves the wafer to two locations that leave the secondregistration mark in the field of view to measure how movements of thescanning arm cause the position of the second registration mark to varythe camera's frame of reference (steps 1502-1508). The program thencalculates the angle of the camera relative to the stepping motor axesfrom the measured positions of the registration marks and the knownchange in position of the scanning arm (step 1509). The wafer is movedby calculating the amount needed to move the wafer to get to the homeposition given the camera angle and the apparent displacement of thesecond registration mark from the home position (step 1510). The programchecks whether the wafer is at the home position (steps 1511-1512). Ifthe wafer is not in the home position, the program repeats steps1510-1512.

[0201]FIG. 18 shows the routine for controlling the treatment in a flowcell, including rinsing and deprotection of a wafer. When the first stepof treatment starts (step 1600), a timer is set for the duration of thetreatment (step 1601). When the time expires, the timer calls thedo_alrm routine which checks whether all the treatment steps are done(step 1603). If so, the do_alrm routine indicates this to the program(step 1604). If not, the next treatment is started (step 1605), and atimer is set for the duration of the treatment as before (step 1606).

[0202]FIG. 19 shows in detail a routine for measuring the centerposition of the first or second registration mark. The program firstobtains from the frame capture circuit a two-dimensional array of pixelsof a digital image taken by the camera (step 1702). Typically, theregistration mark will not be rotated more than one degree or so fromits aligned position. A semi-vertical line and a semi-horizontal linecan be identified from the array of pixels because one of the two linesin the registration mark will appear to be vertical and the other to behorizontal. The program calculates the equation for the semi-verticalline (step 1703). Similarly, the program calculates the equation for thesemi-horizonal line (step 1704). The program then calculates theintersection of the two lines and records the position (step 1705). Ifthe current and previous calculations of the position of theregistration mark agree within some tolerance, the program returns thecalculated position as the center position of the registration mark(steps 1707-1708). If they don't agree or any of the steps requires toestimate the position fails, the program re-tries at step 1701.

[0203]FIG. 20 shows in detail the programming steps for calculating theequation of the semi-vertical line or the semi-horizontal line. First,the pixel values are adjusted against the background (step 1801) bysubtracting the background intensity from each pixel in order tocompensate the effect of different lighting backgrounds. Then, for eachrow or column of the picture (whichever is perpendicular to the expectedline), the program finds the location of the pixel of the maximumintensity (step 1802). The program makes a histogram of the positionscalculated above and discards the positions below an occurrencefrequency cutoff value (step 1803). The program performs a regression onthe remaining points to get the equation for a line (step 1804). Theprogram calculates the standard deviation of the points from theregression line (step 1805). The program throws away those points whosedistance from the regression line is large compared to the standarddeviation calculated in the last step (step 1806). The program performsa regression on the remaining points to get another equation. If thelast two equations calculated agree within a certain tolerance, theprogram returns the equation (steps 1807-1809). The program continuesthe cycle of discarding points and regressing until either successiveequations agree, or too few points remain.

[0204]FIG. 21 shows in detail the programming steps necessary for theDo_a_layer routine that controls the printer head and the scanning armto deposit a particular layer. The scanning arm moves the wafer suchthat the upper right corner of the wafer just overlaps the top row ofthe nozzles in the first inkjet print head (step 1900). The program thenreads in the oligo specification file containing the oligonucleotidesequences for the wafer and extracts the nucleoside specification forthe current layer (step 1901). The program then calculates the entirecontents of the spit vector RAM for this layer from the informationobtained in previous steps (step 1902). The spit vector RAM containsspit vectors representing information of how to fire the array ofnozzles at each trigger point (X-position).

[0205] Once the scanning (deposition) of the current layer has beendone, the chemicals are allowed to dry and the wafer is placed on awafer elevator (steps 1904-1905). If the scanning has not been done, theprogram loads into the spit vector RAM the appropriate portion of thespit vectors from step 1902 for the part of the next layer to scan. Theprogram then loads the trigger RAM with the X-locations calculated forthat wafer during the initialization (step 1907). The wafer is scannedback and forth the appropriate number of times at the appropriateY-locations as calculated in step 1010 (step 1908).

EXAMPLE 1 Comparison of Result of Oligonucleotide Synthesis WithPropylene Carbonate vs. Acetonitrile Solvent

[0206] Nucleoside phosphoramidites used in this experiment are of theEXPEDITE™ type, and were obtained from Perseptive Biosystems,Framingham, Mass. The primary amino groups of the base portion of theadenosine (A), cytidine (C) and guanosine (G) nucleosides were protectedwith t-butylphenoxyacetyl (tBPA) groups. The 5′-hydroxyl groups of theA, C, G and thymidine (T) nucleosides were protected with adimethoxytrityl (DMT) group. The 3′-hydroxyl groups of the A, C, G and Tnucleosides were derivatized asβ-cyanoethyl-N,N-diisopropylphosphoramidites.

[0207] This example compares the efficiency of nucleoside coupling whenpropylene carbonate is used as a reaction solvent, relative to that whenacetonitrile is used, in conventional, solid phase nucleoside synthesis.

[0208] As shown below in Table 1, eight separate oligonucleotidehomopolymers of A, T, C and G, each being eleven nucleotides in length,were assembled using either propylene carbonate or acetonitrile as thereaction solvent. Reagents were dispensed from an Applied Biosystemsmodel 380B synthesizer, a non-inkjet synthesizer, using phosphoramiditechemistry according the manufacturer's instructions. A trityl assay (seeT. Atkinson et al., Solid-Phase Synthesis of Oligodeoxyribonucleotidesby the Phosphite-Triester Method, in Oligonucleotide Synthesis (M. J.Gait ed., 1984)) was used to estimate stepwise yields on all eightsyntheses. This assay measures the amount of dimethoxytrityl groupreleased during the deprotection step of the synthetic cycle. Themeasurement is conveniently carried out photometrically since thedimethoxytrityl group absorbs light strongly at 498 nm. Using thisassay, an estimate of the efficiency of the synthetic reactions was madeby comparing the amounts of dimethoxytrityl released from one cycle tothe next. As shown in Table 1, yields of oligonucleotides that areobtained using either propylene carbonate or acetonitrile solvents, arecomparable. TABLE 1 Assembly of oligonucleotide homopolymers usingacetonitrile or propylene carbonate polydT polydG polydA polydC % yieldA* PC A PC A PC A PC Average 99.4 99.6 99.3 97.4 97.8 96.6 98.9 98.4Overall 88.8 89.4 87.6 74.1 77.6 65.9 88.8 85.4 Stepwise 98.8 98.9 98.797.0 97.5 95.9 98.8 98.4

EXAMPLE 2 Synthesis of Two-Dimensional Oligonucleotide Arrays Using anInklet Print Head

[0209] This example describes the synthesis of a two-dimensional arrayof oligonucleotides using the synthesis system described in Section5.5.1. With respect to the steps involving deposition of reagents usingan inkjet printing head, i.e., those steps not involving oxidizing,rinsing, capping and deprotection, an earlier version of the softwaredescribed in Section 5.5.2 was used.

[0210] The nucleoside phosphoramidites used in this experiment werethose described in Section 6, above.

[0211] An oxidizing solution, that was used to oxidize nucleosidephosphite triesters to nucleoside phosphate triesters, consisted of90.54% (v/v) tetrahydrofuran, 9.05% (v/v) water, 0.41% (v/v) pyridineand 4.3 g/L iodine.

[0212] As mentioned before, inkjet print heads used herein were EPSONSTYLUS COLOR II™ color heads, available from the manufacturer as spareparts, which consist of three banks of twenty nozzles each. All of thenozzles in each bank were connected to a common fluid intake manifold,such that each inkjet print head had three fluid lines connectedthereto. The complete inkjet assembly consisted of two inkjet printheads mounted together, so as to form an assembly of six banks of twentynozzles each.

[0213] Fifty clean, standard, glass microscope slides (25 mm×75 mm) wereused as the substrates upon which the oligonucleotide arrays wereassembled, and were derivatized according to the procedure of E. M.Southern et al., Genomics 13(4):1008-1017 (1992). The slides weresubmerged in a bath of 200 mL of glycidoxypropyltrimethoxysilane, 800 mLof anhydrous xylenes and 10 mL of diisopropylethylamine for 8 h at 80°C. with stirring, and then rinsed with ethanol and dried under nitrogen.The resulting substrates were placed in a bath of 800 mL oftetraethylene glycol and 3 mL of conc. H₂SO₄ for 8 h at 80° C. withstirring, and then rinsed with ethanol and dried under nitrogen.

[0214] Four of the six inkjet banks of the assembly were loaded with 0.1M solutions (propylene carbonate) of each nucleoside phosphoramidite andone of those six inkjet banks was loaded with a 0.5 M solution of5-ethylthiotetrazole in propylene carbonate.

[0215] The derivatized substrate was affixed to an X-Y translation stagethat was driven by two stepping motors via a lead screw. A computer,along with an appropriate electronic interface, was used to synchronizethe firing of the inkjet print head with the motion of the X-Ytranslation stage, so as to deliver one 42 pL drop of the appropriatenucleoside phosphoramidite solution, followed by one 42 pL drop of the5-ethylthiotetrazole solution to each region of the substrate whereoligonucleotide synthesis was to take place. This reaction, whichresulted in the coupling of each nucleoside to the substrate via atetraethyleneglycol linker, was allowed to proceed for 60 seconds undera nitrogen atmosphere. The substrate was rinsed with acetonitrile toremove excess reagents, and dried with anhydrous nitrogen.

[0216] The resulting substrate was submerged in a bath of the oxidizingsolution for 30 seconds so as to convert the resulting nucleosidephosphite triesters to nucleoside phosphate triesters. The substrate wasthen rinsed again with acetonitrile, and then treated with a solution of20 μL of perfluorooctanoyl chloride in 50 mL of anhydrous xylene, so asto cap all of the unreacted hydroxyl groups of the tetraethylene glycolbonded to the substrate.

[0217] The resulting substrate was rinsed with acetonitrile, dried withanhydrous nitrogen, and then dipped for 60 seconds in a solution of 2.5%dichloroacetic acid in dichloromethane which removed the dimethoxytritylprotecting group from the 5′-hydroxyl group of nucleoside. After a finalrinse with acetonitrile, and a drying stream of dry nitrogen, thesubstrate was subjected to 19 iterations of the (a) nucleoside coupling,(b) acetonitrile rinsing, (c) oxidation, (d) acetonitrile rinsing, (e)dimethoxytrityl deprotecting and (f) acetonitrile rinsing steps.

[0218] Finally, the substrate was dipped in undiluted ethanolamine for20 minutes, at room temperature, to remove both the tBPA protectinggroups from the nucleoside bases, and the cyanoethyl groups from thephosphate linkages between adjacent to nucleosides to provide phosphategroups. The substrate was then rinsed with ethanol, and then withacetonitrile, leaving the resulting oligonucleotide attached to thesubstrate.

[0219] The present invention is not to be limited in scope by thespecific embodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention, and any embodimentswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention, in additionto those shown and described herein, will become apparent to thoseskilled in the art, and are intended to fall within the appended claims.

[0220] A number of references have been cited, and the entiredisclosures of which are incorporated herein by reference.

What is claimed is:
 1. A microdroplet of a solution, the solutioncomprising a solvent having a boiling point of 150° C. or above, asurface tension of 30 dynes/cm or above, and a viscosity of 0.015g/(cm)(sec) or above.
 2. The microdroplet of claim 1 , wherein thesolvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 3. The microdropletof claim 2 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 4. Themicrodroplet of claim 2 , wherein said solvent is propylene carbonate.5. The microdroplet of claim 1 , wherein the volume of said microdropletis 100 pL or less.
 6. The microdroplet of claim 5 , wherein the volumeof said microdroplet is 50 pL or less.
 7. The microdroplet of claim 1 ,wherein the solution comprises a nucleoside or activated nucleoside. 8.The microdroplet of claim 7 , wherein the solution comprises anactivated nucleoside that contains an activated phosphorous-containinggroups selected from the group consisting of phosphodiester,phosphotriester, phosphate triester, H-phosphonate and phosphoramiditegroups.
 9. A method for chemical synthesis, comprising the step ofdispensing a microdroplet of a solution comprising (i) a first chemicalspecies, and (ii) a solvent, such that the microdroplet impinges asecond chemical species and the first chemical species reacts with thesecond chemical species to form a third chemical species, the solventhaving a boiling point of 150° C. or above, a surface tension of 30dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above. 10.The method of claim 9 , wherein said solvent has the formula (I):

wherein A=O or S; O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) or CH₂;and R=C₁-C₂₀ straight or branched chain alkyl.
 11. The method of claim10 , wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 12. Themethod of claim 10 , wherein said solvent is propylene carbonate. 13.The method of claim 9 , wherein the volume of said microdroplet is 100pL or less.
 14. The method of claim 13 , wherein the volume of saidmicrodroplet is 50 pL or less.
 15. The method of claim 9 , wherein thethird chemical species is formed in the presence of a catalyst.
 16. Themethod of claim 9 , further comprising the step of dispensing amicrodroplet of a solution comprising (i) a catalyst and (ii) thesolvent, such that the microdroplet comprising the catalyst and solventimpinges the second chemical species subsequent to impingement by themicrodroplet that comprises the first chemical species and solvent. 17.The method of claim 9 , wherein the second chemical species is attachedto a substrate, directly or via a linker molecule.
 18. The method ofclaim 17 , wherein the substrate is selected from the group consistingof glass, silica, silicon, polypropylene, TEFLON®, polyethylimine,nylon, fiberglass, paper and polystyrene.
 19. The method of claim 17 ,wherein the second chemical species is attached to a linker that isattached to the substrate.
 20. The method of claim 9 , wherein the firstchemical species and second chemical species are nucleosides oractivated nucleosides.
 21. The method of claim 20 , wherein the chemicalspecies comprises an activated nucleoside that contains activatedphosphorous-containing groups selected from the group consisting ofphosphodiester, phosphotriester, phosphate triester, H-phosphonate andphosphoramidite groups.
 22. The method of claim 9 , wherein the chemicalbeing synthesized is an oligonucleotide or a peptide.
 23. The method ofclaim 22 , wherein the chemical being synthesized is anoligodeoxyribonucleotide or an oligoribonucleotide.
 24. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving a phosphoramidite group at its 3′ position, and a protectinggroup at its 5′ position and (ii) a solvent having a boiling point of150° C. or above, a surface tension of 30 dynes/cm or above, and aviscosity of 0.015 g/(cm)(sec) or above, such that the microdropletimpinges a substrate with a linker attached thereto having hydroxylgroups, and forms a microdot upon the substrate; (b) dispensing a secondmicrodroplet of a solution comprising (i) a catalyst and (ii) thesolvent, such that the second microdroplet impinges the microdot andfacilitates a reaction between the phosphoramidite group of the firstnucleoside and an hydroxyl group of the linker, resulting in theconversion of the phosphoramidite group to a 3′ phosphite group, and thepresence of unreacted hydroxyl groups of the linker; (c) washing thesubstrate with an oxidizing agent to convert the 3′ phosphite group to a3′ phosphate group; (d) rinsing the substrate with a deprotecting agentwhich removes the protecting group from the 5′ position of the firstnucleoside, and yields a 5′ hydroxyl group; (e) dispensing a thirdmicrodroplet of a solution comprising (i) a second nucleoside having aphosphoramidite group at its 3′ position, and a protecting group at its5′ position and (ii) the solvent, such that the second microdropletimpinges the microdot; and (f) dispensing a fourth microdroplet of asolution comprising (i) the catalyst and (ii) the solvent, such that thefourth microdroplet impinges the microdot and facilitates a reactionbetween the phosphoramidite group of the second nucleoside and the 5′hydroxyl group of the first nucleoside, resulting in the coupling of thesecond nucleoside to the first nucleoside.
 25. The method of claim 24further comprising the steps of performing successive iterations ofsteps (c)-(f).
 26. The method of claim 24 , further comprising afterstep (c) and before step (d) the step of treating the substrate with acapping reagent which caps the unreacted hydroxyl groups of the linker.27. The method of claim 26 , wherein the capping reagent isperfluorooctanoyl chloride.
 28. The method of claim 24 wherein saidsolvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 29. The method ofclaim 28 , wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 30. Themethod of claim 28 , wherein said solvent is propylene carbonate. 31.The method of claim 24 , wherein the substrate is glass.
 32. The methodof claim 24 , wherein the catalyst is 5-ethylthiotetrazole.
 33. Themethod of claim 24 , wherein the oxidizing agent is a solutioncomprising iodine and water.
 34. An array of microdots on a substrate,said microdots comprising (i) a chemical species, and (ii) a solventhaving a boiling point of 150° C. or above, a surface tension of 30dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above. 35.The array of claim 34 , wherein said solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 36. The array ofclaim 35 , wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 37. Thearray of claim 35 , wherein said solvent is propylene carbonate.
 38. Thearray of claim 34 , wherein each of said microdots has a diameter in therange of 1 to 1000 μm.
 39. The array of claim 38 , wherein each of saidmicrodots has a diameter in the range of 10 to 500 μm.
 40. The array ofclaim 39 , wherein each of said microdots has a diameter in the range of40 to 100 μm.
 41. The array of claim 34 , wherein the chemical speciesis an oligonucleotide or a peptide.
 42. The array of claim 41 , whereinthe chemical species is an oligodeoxyribonucleotide or anoligoribonucleotide.
 43. The array of claim 34 , wherein the microdotsare separated from each other by hydrophobic domains.
 44. An automatedsystem comprising: an inkjet print head for spraying a microdropletcomprising a chemical species on a substrate; a scanning transport forscanning the substrate adjacent to the print head to selectively depositthe microdroplet at specified sites; a flow cell for treating thesubstrate on which the microdroplet is deposited by exposing thesubstrate to one or more selected fluids; a treating transport formoving the substrate between the print head and the flow cell fortreatment in the flow cell; and an alignment unit for aligning thesubstrate correctly relative to the print head each time when thesubstrate is positioned adjacent to the print head for deposition. 45.The system of claim 44 , wherein the inkjet printhead contains asolution comprising the chemical species dissolved in a solvent.
 46. Thesystem of claim 45 , wherein the chemical species is a monomer unit of abiopolymer.
 47. The system of claim 45 , wherein the solution furthercomprises a catalyst.
 48. The system of claim 45 wherein the solutioncomprises a solvent having a boiling point of 150° C. or above, asurface tension of 30 dynes/cm or above, and a viscosity of 0.015g/(cm)(sec) or above.
 49. The system of claim 48 , wherein the solventhas the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄) or CH₂; andR=C₁-C₂₀ straight or branched chain alkyl.
 50. The system of claim 49 ,wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 51. Thesystem of claim 48 , wherein said solvent is propylene carbonate. 52.The system of claim 46 which is for synthesizing an oligonucleotide, andwherein the monomer is a nucleoside or nucleoside derivative.
 53. Thesystem of claim 52 , wherein the nucleoside is a deoxyribonucleoside ora ribonucleoside.
 54. The system of claim 44 , wherein the inkjet printhead comprises an array of piezoelectric pumps.
 55. The system of claim54 , further comprising an external reservoir connected to supply thechemical species to the print head.
 56. The system of claim 55 , whereinthe external reservoir contains a solution comprising the chemicalspecies dissolved in a solvent.
 57. The system of claim 56 , wherein thesolvent has a boiling point of 150° C. or above, a surface tension of 30dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above. 58.The system of claim 57 , wherein the solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 59. The system ofclaim 58 , wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 60. Thesystem of claim 58 , wherein said solvent is propylene carbonate.
 61. Anautomated system for synthesizing oligonucleotides on a substrate,comprising: an inkjet print head for spraying a solution comprising anucleoside or activated nucleoside on a substrate; a scanning transportfor scanning the substrate adjacent to the print head to selectivelydeposit the nucleoside at specified sites; a flow cell for treating thesubstrate on which the monomer is deposited by exposing the substrate toone or more selected fluids; a treating transport for moving thesubstrate between the print head and the flow cell for treatment in theflow cell; and an alignment unit for aligning the substrate correctlyrelative to the print head each time when the substrate is positionedadjacent to the print head for deposition.
 62. The system of claim 61 ,wherein the inkjet print head comprises an array of piezoelectric pumps.63. The system of claim 61 , further comprising: an external reservoirconnected to supply the nucleoside to the print head.
 64. The system ofclaim 63 , wherein the external reservoir contains a solution comprisingsaid nucleoside or activated nucleoside dissolved in propylenecarbonate.
 65. The system of claim 61 , further comprising a pluralityof external reservoirs connected to the printer head, each externalreservoir storing a nucleoside or activated nucleoside.
 66. The systemof claim 61 , further comprising control logic configured to perform thefollowing steps: moving the substrate over the print head with thescanning transport; firing the print head repeatedly to deposit thenucleoside or activated nucleoside monomer at the specified loci on thesubstrate; and transferring the substrate to the flow cell with thetreating transport.
 67. The system of claim 61 , wherein the scanningtransport comprises: a vacuum chuck for holding the substrate; and atranslational stage connected to move the vacuum chuck with respect tothe print head.
 68. The system of claim 67 , wherein the vacuum chuck isrotatable for alignment with the print head.
 69. The system of claim 68, wherein the vacuum chuck is engageable by a stationary element torotate the vacuum chuck for alignment with the print head.
 70. Thesystem of claim 67 , wherein the translational stage is driven bymotorized means.
 71. The system of claim 70 , wherein the motorizedmeans is a stepping motor.
 72. The system of claim 61 , wherein the flowcell has means for rinsing off unconnected monomers.
 73. The system ofclaim 61 , wherein the treating transport comprises: a vacuum chuck forholding the substrate; and a translational stage connected to move thesecond vacuum chuck and to move the substrate to and from the flow cell.74. The system of claim 73 , wherein the translational stage is drivenby motorized means.
 75. The system of claim 74 , wherein the motorizedmeans is a stepping motor.
 76. The system of claim 61 , wherein saidalignment unit comprises a camera positioned adjacent to the substrateto positionally calibrate the substrate.
 77. The system of claim 61 ,wherein said alignment unit comprises a marker that can be activated toestablish one or more marks at particular loci on the substrate forpositionally calibrating the substrate.
 78. The system of claim 61 ,wherein said alignment unit comprises: a marker that can be activated toestablish one or more marks at particular loci on the substrate; and acamera positioned adjacent to the substrate to located said marksrelative to the printer head.
 79. The system of claim 78 , furthercomprising a tip that can be activated to scratch marks at particularloci on the substrate for positionally calibrating the substrate. 80.The system of claim 61 , wherein said alignment unit comprises: controllogic connected to control the movement of the scanning transport; amarker that can be activated to establish one or more marks atparticular loci on the substrate; and a camera positioned adjacent tothe substrate to locate said marks relative to the printer head.
 81. Thesystem of claim 80 , wherein the control logic is configured to performthe following steps: moving the substrate over the marker to establishone or more marks on the substrate; subsequently locating the marks withthe camera; determining the position of the substrate with respect tothe print head with reference to the marks; and calibrating the scanningtransport in response to the determined position of the substrate withrespect to the printer head.
 82. The system of claim 80 , furthercomprising a stationary element that engages the substrate chuck torotate the substrate chuck for alignment with the print head, whereinthe control logic is configured to perform the following steps: movingthe substrate over the marker to establish one or more marks on thesubstrate; subsequently locating the marks with the camera; determiningmisalignment of the substrate relative to the print head with referenceto the marks; and moving the translational stage to (a) engage thesubstrate chuck with the stationary element, and (b) rotate thesubstrate chuck by an angular displacement that corrects for themisalignment.
 83. The system of claim 61 , further comprising a transferstation that supports the substrate for transfer between the treatingtransport and the scanning transport.
 84. A method of controlling asystem synthesizing a biopolymer on a substrate using a computer havinga memory for storing a control program and data, wherein the system hasan inkjet print head for spraying a microdroplet on the substrate, ascanning transport for scanning the substrate adjacent to the print headto selectively deposit the microdroplet, an alignment unit for detectingmisalignment of the substrate with respect to the print head at eachdeposition step, a flow cell for treating the substrate, and a treatingtransport for moving the substrate between the printer head and the flowcell, the method comprising the steps of: (a) aligning the substraterelative to the print head by processing data from the alignment unitand by sending a signal to the scanning transport to move the substrateso as to correct misalignment of the substrate; (b) selectivelydepositing a microdroplet on the substrate by sending a signal to theprint head to spray the microdroplet and by sending a signal to thescanning transport to move the substrate adjacent to the print head sothat the microdroplet can be deposited at specified loci on thesubstrate; and (c) controlling treatment of the substrate by sending asignal to the treating transport to move the substrate to the flow celland by sending a signal to the flow cell to control operation of theflow cell.
 85. The method of claim 84 , wherein the microdropletcomprises a monomer unit of a biopolymer.
 86. The method of claim 85 ,wherein the microdroplet further comprises a catalyst.
 87. The method ofclaim 84 , wherein the microdroplet comprises a solvent.
 88. The methodof claim 87 , wherein the solvent has a boiling point of 150° C. orabove, a surface tension of 30 dynes/cm or above, and a viscosity of0.015 g/(cm)(sec) or above.
 89. The method of claim 88 , wherein thesolvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 90. The method ofclaim 89 , wherein the solvent is selected from the group consisting of:N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 91. Themethod of claim 87 , wherein said solvent is propylene carbonate. 92.The method of claim 84 , further comprising the steps of repeating saidsteps (a)-(c).
 93. The method of claim 85 , further comprising the stepsof repeating said steps (a)-(c) to form a two-dimensional biopolymerarray.
 94. The method of claim 84 , wherein the biopolymer is anoligonucleotide.
 95. The method of claim 94 , wherein theoligonucleotide is an oligodeoxyribonucleotide or anoligoribonucleotide.
 96. The method of claim 85 , further comprisingrepeating said steps (a)-(c) to form a two-dimensional oligonucleotidearray.
 97. The method of claim 84 , wherein the step of aligning thesubstrate comprises the steps of: (a) moving the substrate over a markerto establish one or marks on the substrate; (b) subsequently locatingthe marks with a camera; (c) determining misalignment of the substraterelative to the print head with reference to the marks; and (d) movingthe substrate to correct the misalignment.
 98. The method of claim 97 ,wherein the step of moving the substrate to correct the misalignment isdone by moving the substrate in a linear motion in X and Y directionsand by rotating the substrate.
 99. A method of controlling a systemsynthesizing oligonucleotides on a substrate using a computer having amemory for storing a control program and data, wherein the system has aninkjet print head for spraying a solution comprising a nucleoside oractivated nucleoside, on the substrate, a scanning transport forscanning the substrate adjacent to the print head to selectively depositthe solution, an alignment unit for detecting misalignment of thesubstrate with respect to the print head at each deposition step, a flowcell for treating the substrate, and a treating transport for moving thesubstrate between the printer head and the flow cell, the methodcomprising the steps of: (a) placing the substrate with respect to theprint head and establishing marks on the substrate; (b) selectivelydepositing the solution on the substrate by sending a signal to theprint head to spray the solution and by sending a signal to the scanningtransport to move the substrate adjacent to the print head so that thesolution can be deposited at specified loci on the substrate; (c)controlling treatment of the substrate by sending a signal to thetreating transport to move the substrate to the flow cell and by sendinga signal to the flow cell to control operation of the flow cell; and (d)placing the substrate adjacent to the print head and aligning thesubstrate with respect to the print head by processing data from thealignment unit and by sending a signal to the scanning transport to movethe substrate so as to correct misalignment of the substrate.
 100. Themethod of claim 99 , further comprising repeating said steps (a)-(d) toform a two-dimensional oligonucleotide array.
 101. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving an activated O-succinate group at its 3′ position, and aprotecting group at its 5′ position and (ii) a solvent having a boilingpoint of 150° C. or above, a surface tension of 30 dynes/cm or above,and a viscosity of 0.015 g/(cm)(sec) or above, such that themicrodroplet impinges a substrate with a linker attached thereto havingan amine group, and forms a microdot upon the substrate; (b) rinsing thesubstrate with a deprotecting agent which removes the protecting groupfrom the 5′ position of the first nucleoside, and exposes a 5′ hydroxylgroup; (c) dispensing a second microdroplet of a solution comprising (i)a second nucleoside having a phosphoramidite group, containing acyanoethyl group, at its 3′ position, and a protecting group at its 5′position and (ii) the solvent, such that the second microdropletimpinges the microdot; (d) dispensing a third microdroplet of a solutioncomprising (i) a catalyst and (ii) the solvent, such that the thirdmicrodroplet impinges the microdot and facilitates a reaction betweenthe phosphoramidite group of the second nucleoside and the 5′ hydroxylgroup of the first nucleoside, resulting in the conversion of thephosphoramidite group to a phosphite group; (e) washing the substratewith an oxidizing agent to convert the 3′ phosphite group to a 3′phosphate group; (f) performing successive iterations of steps (b)-(e);(g) treating the product of step (f) with a second deprotecting agentthat converts the cyanoethylphosphate groups, of the product of step(f), to phosphate groups; and (h) treating the product of step (g) witha hydrolyzing agent which cleaves the oligonucleotide from the linker.102. The method of claim 101 wherein said solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 103. The method ofclaim 101 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 104. Themethod of claim 101 , wherein said solvent is propylene carbonate. 105.The method of claim 101 , wherein the substrate is glass.
 106. Themethod of claim 101 , wherein the catalyst is 5-ethylthiotetrazole. 107.The method of claim 101 , wherein the oxidizing agent is a solutioncomprising iodine and water.
 108. The method of claim 101 , wherein thehydrolyzing agent is selected from the group consisting of hydroxideion, CH₃NH₂ and concentrated aqueous NH₄OH.
 109. An automated system forsynthesizing oligonucleotides on a substrate, comprising: an inkjetprint head having an array of pumps for depositing a nucleoside oractivated nucleoside at specified loci on the substrate; a firsttranslational stage having at least two axes of movement; a firstsubstrate chuck mounted for movement by the translational stage, thefirst substrate chuck being adapted to hold the substrate and moveadjacent to the print head; a flow cell that receives the substrate andthat exposes the substrate to one or more selected fluids; a secondtranslational stage that has at least two axes of movement; a secondsubstrate chuck mounted for movement by the second translation stage,the second substrate chuck being adapted to hold the substrate and movebetween the print head and the flow cell; control logic connected tocontrol movement of the first and second translational stages; a markerpositioned adjacent to the print head that can be activated to markparticular loci on the substrate for positionally calibrating thesubstrate with respect to the print head; and a camera positionedadjacent to the print head to positionally calibrate the substrate withrespect to the print head, wherein the camera is connected to provideimages to the control logic.
 110. The system of claim 109 , wherein thefirst and the second translational stages are driven by stepping motors.111. The system of claim 109 , wherein the first and second substratechucks are vacuum chucks.
 112. The system of claim 109 , wherein thefirst substrate chuck is rotatable for alignment with the print head.113. The system of claim 109 , further comprising a tip that can beactivated to scratch marks at particular loci on the substrate forpositionally calibrating the substrate.
 114. The system of claim 109 ,further comprising: a reservoir connected to the print head to supplythe nucleoside or activated nucleoside to the print head, wherein thereservoir contains said nucleoside or activated nucleoside dissolved ina solvent.
 115. The system of claim 114 , wherein the solvent has aboiling point of 150° C. or above, a surface tension of 30 dynes/cm orabove, and a viscosity of 0.015 g/(cm)(sec) or above.
 116. The system ofclaim 115 , wherein the solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₂-C₂₀ straight or branched chain alkyl.
 117. The system ofclaim 116 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 118. Thesystem of claim 115 , wherein said solvent is propylene carbonate. 119.A solution comprising a solvent and a nucleoside or activatednucleoside, said solvent having a boiling point of 150° C. or above, asurface tension of 30 dynes/cm or above, and a viscosity of 0.015g/(cm)(sec) or above.
 120. The solution of claim 119 , wherein thesolvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₂-C₂₀ straight or branched chain alkyl.
 121. The solution ofclaim 120 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 122. Thesolution of claim 119 , wherein said solvent is propylene carbonate.123. The solution of claim 119 , wherein the solution comprises anactivated nucleoside that contains an activated phosphorous-containinggroup selected from the group consisting of phosphodiester,phosphotriester, phosphate triester, H-phosphonate and phosphoramiditegroup.
 124. An apparatus programmed for controlling a systemsynthesizing a biopolymer on a substrate, wherein the system has aninkjet print head for spraying a microdroplet on the substrate, ascanning transport for scanning the substrate adjacent to the print headto selectively deposit the microdroplet, an alignment unit for detectingmisalignment of the substrate with respect to the print head at eachdeposition step, a flow cell for treating the substrate, and a treatingtransport for moving the substrate between the printer head and the flowcell, the controller comprising: (a) means for controlling alignment ofthe substrate relative to the print head by processing data from thealignment unit and by sending a signal to the scanning transport to movethe substrate so as to correct misalignment of the substrate; (b) meansfor controlling selective deposition of a microdroplet on the substrateby sending a signal to the print head to spray the microdroplet and bysending a signal to the scanning transport to move the substrateadjacent to the print head so that the microdroplet can be deposited atspecified loci on the substrate; and (c) means for controlling treatmentof the substrate by sending a signal to the treating transport to movethe substrate to the flow cell and by sending a signal to the flow cellto control operation of the flow cell.
 125. The apparatus of claim 124 ,wherein the inkjet print head contains a solution comprising a monomerunit of a biopolymer.
 126. The apparatus of claim 125 , wherein thesolution further comprises a catalyst.
 127. The apparatus of claim 125 ,wherein the solution comprises a solvent that has a boiling point of150° C. or above, a surface tension of 30 dynes/cm or above, and aviscosity of 0.015 g/(cm)(sec) or above.
 128. The apparatus of claim 127, wherein the solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 129. The apparatusof claim 124 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 130. Theapparatus of claim 127 , wherein said solvent is propylene carbonate.131. An apparatus programmed for controlling a system synthesizing abiopolymer on a substrate, wherein the system has an inkjet print headfor spraying a microdroplet on the substrate, a scanning transport forscanning the substrate adjacent to the print head to selectively depositthe microdroplet, an alignment unit for detecting misalignment of thesubstrate with respect to the print head at each deposition step, a flowcell for treating the substrate, and a treating transport for moving thesubstrate between the printer head and the flow cell, said apparatuscomprising one or more computer systems programmed for: (a) controllingalignment of the substrate relative to the print head by processing datafrom the alignment unit and by sending a signal to the scanningtransport to move the substrate so as to correct misalignment of thesubstrate; (b) controlling selective deposition of a microdroplet on thesubstrate by sending a signal to the print head to spray themicrodroplet and by sending a signal to the scanning transport to movethe substrate adjacent to the print head so that the microdroplet can bedeposited at specified loci on the substrate; and (c) controllingtreatment of the substrate by sending a signal to the treating transportto move the substrate to the flow cell and by sending a signal to theflow cell to control operation of the flow cell.
 132. The apparatus ofclaim 131 , further comprising a reservoir connected to the print headto suppy a nucleoside or activated nucleoside to the print head, whereinthe reservoir contains said nucleoside or activated nucleoside dissolvedin a solvent.
 133. The apparatus of claim 131 , wherein the inkjet printhead contains a solution comprising a monomer unit of a biopolymer. 134.The apparatus of claim 133 , wherein the solution further comprises acatalyst.
 135. The apparatus of claim 133 , wherein the solutioncomprises a solvent that has a boiling point of 150° C. or above, asurface tension of 30 dynes/cm or above, and a viscosity of 0.015g/(cm)(sec) or above.
 136. The apparatus of claim 135 , wherein thesolvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.
 137. The apparatusof claim 136 , wherein the solvent is selected from the group consistingof: N-methyl-2-pyrrolidone; 2-pyrrolidone; propylene carbonate;γ-valerolactone; 6-caprolactam; ethylene carbonate; γ-butyrolactone;δ-valerolactone; 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;ethylene trithiocarbonate; and 1,3-dimethyl-2-imidazolidinone.
 138. Theapparatus of claim 135 , wherein said solvent is propylene carbonate.139. An inkjet print head containing the solution of claim 119 .
 140. Aninkjet print head containing the solution of claim 120 .
 141. An inkjetprint head containing the solution of claim 121 .
 142. An inkjet printhead containing the solution of claim 122 .
 143. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving a phosphoramidite group at its 5′ position, and a protectinggroup at its 3′ position and (ii) a solvent having a boiling point of150° C. or above, a surface tension of 30 dynes/cm or above, and aviscosity of 0.015 g/(cm)(sec) or above, such that the microdropletimpinges a substrate with a linker attached thereto having hydroxylgroups, and forms a microdot upon the substrate; (b) dispensing a secondmicrodroplet of a solution comprising (i) a catalyst and (ii) thesolvent, such that the second microdroplet impinges the microdot andfacilitates a reaction between the phosphoramidite group of the firstnucleoside and an hydroxyl group of the linker, resulting in theconversion of the phosphoramidite group to a 5′ phosphite group, and thepresence of unreacted hydroxyl groups of the linker; (c) washing thesubstrate with an oxidizing agent to convert the 5′ phosphite group to a5′ phosphate group; (d) rinsing the substrate with a deprotecting agentwhich removes the protecting group from the 3′ position of the firstnucleoside, and yields a 3′ hydroxyl group; (e) dispensing a thirdmicrodroplet of a solution comprising (i) a second nucleoside having aphosphoramidite group at its 5′ position, and a protecting group at its3′ position and (ii) the solvent, such that the second microdropletimpinges the microdot; and (f) dispensing a fourth microdroplet of asolution comprising (i) the catalyst and (ii) the solvent, such that thefourth microdroplet impinges the microdot and facilitates a reactionbetween the phosphoramidite group of the second nucleoside and the 3′hydroxyl group of the first nucleoside, resulting in the coupling of thesecond nucleoside to the first nucleoside.
 144. The method of claim 143, further comprising after step (c) and before step (d) the step oftreating the substrate with a capping reagent which caps the unreactedhydroxyl groups of the linker.
 145. The method of claim 144 , whereinthe capping reagent is perfluorooctanoyl chloride.
 146. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving an activated O-succinate group at its 5′ position, and aprotecting group at its 3′ position and (ii) a solvent having a boilingpoint of 150° C. or above, a surface tension of 30 dynes/cm or above,and a viscosity of 0.015 g/(cm)(sec) or above, such that themicrodroplet impinges a substrate with a linker attached thereto havingan amine group, and forms a microdot upon the substrate; (b) rinsing thesubstrate with a deprotecting agent which removes the protecting groupfrom the 3′ position of the first nucleoside, and exposes a 3′ hydroxylgroup; (c) dispensing a second microdroplet of a solution comprising (i)a second nucleoside having a phosphoramidite group containing acyanoethyl group, at its 5′ position, and a protecting group at its 3′position and (ii) the solvent, such that the second microdropletimpinges the microdot; (d) dispensing a third microdroplet of a solutioncomprising (i) a catalyst and (ii) the solvent, such that the thirdmicrodroplet impinges the microdot and facilitates a reaction betweenthe phosphoramidite group of the second nucleoside and the 3′ hydroxylgroup of the first nucleoside, resulting in the conversion of thephosphoramidite group to a phosphite group; (e) washing the substratewith an oxidizing agent to convert the 5′ phosphite group to a 5′phosphate group; (f) performing successive iterations of steps (b)-(d);(g) treating the product of step (f) with a second deprotecting agentthat converts the cyanoethyl groups, of the product of step (f), tophosphate groups; and (h) treating the product of step (g) with ahydrolyzing agent which cleaves the oligonucleotide from the linker.147. A method for synthesizing an oligonucleotide, comprising the stepsof: (a) dispensing a first microdroplet of a solution comprising (i) afirst nucleoside having a phosphoramidite group at its 3′ position, anda protecting group at its 5′ position and (ii) a solvent having aboiling point of 150° C. or above, a surface tension of 30 dynes/cm orabove, and a viscosity of 0.015 g/(cm)(sec) or above, such that themicrodroplet impinges a substrate with a linker attached thereto havinghydroxyl groups, and forms a microdot upon the substrate; (b) dispensinga second microdroplet of a solution comprising (i) a catalyst and (ii)the solvent, such that the second microdroplet impinges the microdot andfacilitates a reaction between the phosphoramidite group of the firstnucleoside and an hydroxyl group of the linker, resulting in theconversion of the phosphoramidite group to a 3′ phosphite group, and thepresence of unreacted hydroxyl groups of the linker; (c) washing thesubstrate with an oxidizing agent to convert the 3′ phosphite group to a3′ phosphate group; (d) rinsing the substrate with a deprotecting agentwhich removes the protecting group from the 5′ position of the firstnucleoside, and yields a 5′ hydroxyl group; (e) dispensing a thirdmicrodroplet of a solution comprising (i) a second nucleoside having aphosphoramidite group at its 5′ position, and a protecting group at its3′ position and (ii) the solvent, such that the second microdropletimpinges the microdot; and (f) dispensing a fourth microdroplet of asolution comprising (i) the catalyst and (ii) the solvent, such that thefourth microdroplet impinges the microdot and facilitates a reactionbetween the phosphoramidite group of the second nucleoside and the 5′hydroxyl group of the first nucleoside, resulting in the coupling of thesecond nucleoside to the first nucleoside.
 148. The method of claim 147, further comprising after step (c) and before step (d) the step oftreating the substrate with a capping reagent which caps the unreactedhydroxyl groups of the linker.
 149. The method of claim 148 , whereinthe capping reagent is perfluorooctanoyl chloride.
 150. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving a phosphoramidite group at its 5′ position, and a protectinggroup at its 3′ position and (ii) a solvent having a boiling point of150° C. or above, a surface tension of 30 dynes/cm or above, and aviscosity of 0.015 g/(cm)(sec) or above, such that the microdropletimpinges a substrate with a linker attached thereto having hydroxylgroups, and forms a microdot upon the substrate; (b) dispensing a secondmicrodroplet of a solution comprising (i) a catalyst and (ii) thesolvent, such that the second microdroplet impinges the microdot andfacilitates a reaction between the phosphoramidite group of the firstnucleoside and an hydroxyl group of the linker, resulting in theconversion of the phosphoramidite group to a 5′ phosphite group, and thepresence of unreacted hydroxyl groups of the linker; (c) washing thesubstrate with an oxidizing agent to convert the 5′ phosphite group to a5′ phosphate group; (d) rinsing the substrate with a deprotecting agentwhich removes the protecting group from the 3′ position of the firstnucleoside, and yields a 3′ hydroxyl group; (e) dispensing a thirdmicrodroplet of a solution comprising (i) a second nucleoside having aphosphoramidite group at its 3′ position, and a protecting group at its5′ position and (ii) the solvent, such that the second microdropletimpinges the microdot; and (f) dispensing a fourth microdroplet of asolution comprising (i) the catalyst and (ii) the solvent, such that thefourth microdroplet impinges the microdot and facilitates a reactionbetween the phosphoramidite group of the second nucleoside and the 3′hydroxyl group of the first nucleoside, resulting in the coupling of thesecond nucleoside to the first nucleoside.
 151. The method of claim 150, further comprising after step (c) and before step (d) the step oftreating the substrate with a capping reagent which caps the unreactedhydroxyl groups of the linker.
 152. The method of claim 151 , whereinthe capping reagent is perfluorooctanoyl chloride.
 153. A method forsynthesizing an oligonucleotide, comprising the steps of: (a) dispensinga first microdroplet of a solution comprising (i) a first nucleosidehaving an activated O-succinate group at its 5′ position, and aprotecting group at its 3′ position and (ii) a solvent having a boilingpoint of 150° C. or above, a surface tension of 30 dynes/cm or above,and a viscosity of 0.015 g/(cm)(sec) or above, such that themicrodroplet impinges a substrate with a linker attached thereto havingan amine group, and forms a microdot upon the substrate; (b) rinsing thesubstrate with a deprotecting agent which removes the protecting groupfrom the 3′ position of the first nucleoside, and exposes a 3′ hydroxylgroup; (c) dispensing a second microdroplet of a solution comprising (i)a second nucleoside having a phosphoramidite group containing acyanoethyl group, at its 3′ position, and a protecting group at its 5′position and (ii) the solvent, such that the second microdropletimpinges the microdot; (d) dispensing a third microdroplet of a solutioncomprising (i) a catalyst and (ii) the solvent, such that the thirdmicrodroplet impinges the microdot and facilitates a reaction betweenthe phosphoramidite group of the second nucleoside and the 3′ hydroxylgroup of the first nucleoside, resulting in the conversion of thephosphoramidite group to a phosphite group; (e) washing the substratewith an oxidizing agent to convert the 5′ phosphite group to a 5′phosphate group; (f) performing successive iterations of steps (b)-(d);(g) treating the product of step (f) with a second deprotecting agentthat converts the cyanoethyl groups, of the product of step (f), tophosphate groups; and (h) treating the product of step (g) with ahydrolyzing agent which cleaves the oligonucleotide from the linker.154. A method for synthesizing an oligonucleotide, comprising the stepsof: (a) dispensing a first microdroplet of a solution comprising (i) afirst nucleoside having an activated O-succinate group at its 3′position, and a protecting group at its 5′ position and (ii) a solventhaving a boiling point of 150° C. or above, a surface tension of 30dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, suchthat the microdroplet impinges a substrate with a linker attachedthereto having an amine group, and forms a microdot upon the substrate;(b) rinsing the substrate with a deprotecting agent which removes theprotecting group from the 5′ position of the first nucleoside, andexposes a 5′ hydroxyl group; (c) dispensing a second microdroplet of asolution comprising (i) a second nucleoside having a phosphoramiditegroup, having a cyanoethyl group, at its 5′ position, and a protectinggroup at its 3′ position and (ii) the solvent, such that the secondmicrodroplet impinges the microdot; (d) dispensing a third microdropletof a solution comprising (i) a catalyst and (ii) the solvent, such thatthe third microdroplet impinges the microdot and facilitates a reactionbetween the phosphoramidite group of the second nucleoside and the 5′hydroxyl group of the first nucleoside, resulting in the conversion ofthe phosphoramidite group to a phosphite group; (e) washing thesubstrate with an oxidizing agent to convert the 3′ phosphite group to a3′ phosphate group; (f) performing successive iterations of steps(b)-(d); (g) treating the product of step (f) with a second deprotectingagent that converts the cyanoethylphosphate groups, of the product ofstep (f), to phosphate groups; and (h) treating the product of step (g)with a hydrolyzing agent which cleaves the oligonucleotide from thelinker.
 155. The method of any one of claims 143, 146, 147, 150, 153, or154, wherein said solvent has the formula (I):

wherein A=O or S; X=O, S or N(C₁-C₄ alkyl); Y=O, S, N(C₁-C₄ alkyl) orCH₂; and R=C₁-C₂₀ straight or branched chain alkyl.